Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry

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This article details the validation of this antibody for immunohistochemistry of formalin-fixed paraffin embedded murine tissue using a Leica BondMax immunostainer. Using Western blot and IHC on murine wild-type and knockout tissue we have demonstrated that this antibody to ARID1A correctly stains murine tissue by immunohistochemistry." } { "@context": "http://schema.org", "@type": "BreadcrumbList", "itemListElement": [ { "@type": "ListItem", "position": "1", "item": { "@id": "https://f1000research.com/", "name": "Home" } }, { "@type": "ListItem", "position": "2", "item": { "@id": "https://f1000research.com/browse/articles", "name": "Browse" } }, { "@type": "ListItem", "position": "3", "item": { "@id": "https://f1000research.com/articles/3-244/v3", "name": "Application of ARID1A to murine formalin-fixed paraffin embedded tissue..." } } ] } Home Browse Application of ARID1A to murine formalin-fixed paraffin embedded tissue... ALL Metrics - Views Downloads Get PDF Get XML Cite How to cite this article Howat W, Miller J and Gounaris I. Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.12688/f1000research.5514.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. Close Copy Citation Details Export Export Citation Sciwheel EndNote Ref. Manager Bibtex ProCite Sente EXPORT Select a format first Track Share ▬ ✚ Antibody Validation Article Revised Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] Will Howat 1 , Jodi Miller 1 , Ioannis Gounaris 1 Will Howat 1 , Jodi Miller 1 , Ioannis Gounaris 1 PUBLISHED 04 Jun 2015 Author details Author details 1 Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK OPEN PEER REVIEW DETAILS REVIEWER STATUS This article is included in the Antibody Validations gateway. Abstract ARID1A is a known suppressor of tumour formation and the Human Protein Atlas antibody HPA005456 has been demonstrated in previous literature to stain tumour tissue by immunohistochemistry (IHC) in formalin-fixed paraffin embedded human tissue and human cell lines. This article details the validation of this antibody for immunohistochemistry of formalin-fixed paraffin embedded murine tissue using a Leica BondMax immunostainer. Using Western blot and IHC on murine wild-type and knockout tissue we have demonstrated that this antibody to ARID1A correctly stains murine tissue by immunohistochemistry. READ ALL READ LESS Keywords immunohistochemistry, antibody, ARID1A, tissue Corresponding Author(s) Will Howat ( [email protected] ) Close Corresponding author: Will Howat Competing interests: No competing interests were disclosed. Grant information: WH and JM are funded by Cancer Research UK, IG is funded by an MRC Clinical Research Training Fellowship, grant number G1001957. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Copyright: © 2015 Howat W et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). How to cite: Howat W, Miller J and Gounaris I. Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.12688/f1000research.5514.3 ) First published: 15 Oct 2014, 3 :244 ( https://doi.org/10.12688/f1000research.5514.1 ) Latest published: 04 Jun 2015, 3 :244 ( https://doi.org/10.12688/f1000research.5514.3 ) Revised Amendments from Version 2 The article type has been changed from 'Research Note' to 'Antibody Validation Article' to better reflect the type of study presented. The article type has been changed from 'Research Note' to 'Antibody Validation Article' to better reflect the type of study presented. See the authors' detailed response to the review by Stephen McQuaid See the authors' detailed response to the review by Andrew D. Chalmers READ REVIEWER RESPONSES Introduction ARID1A (AT-rich interactive domain 1a) is a member of the SWI/SNF family and its loss has been implicated as a factor in multiple premalignant and malignant conditions, including Barrett’s oesophagus and oesophageal carcinoma as well as endometrial and clear cell ovarian carcinomas and their precursor endometriotic lesions 1 – 4 . The ARID1A antibody from Human Protein Atlas is a rabbit antibody generated against a PrEST (Protein Epitope Signature Tag) fragment of the ARID1A gene and affinity purified against the same fragment 5 . It is thus designated as being “mono-specific” in that the affinity purification removes any non-specific or low affinity binders to the peptide. Through the Human Protein Atlas, the antibody has been tested on a wide variety of human tissue types and human malignancies, as well as for expression in immunofluorescence on U-2 OS, A-431 and U-251 MG cell lines. This demonstrates a nuclear expression in all cell lines and in the majority of tissue types 6 . However, the Western blot data were not supportive and did not produce staining corresponding to the expected size, although the data from a protein array did confirm a peak at the expected size. The antibody has been used to stain human colorectal cancers 7 , clear cell carcinomas 8 and on a variety of clear cell cancer cell lines 9 by immunohistochemistry. To our knowledge, whilst the sequence homology between mouse and human ARID1A is 95%, this antibody has not been qualified using knockout tissue and has not been tested or published on murine tissue and this work represents the first data to do so. Materials and methods Reagent details Details of all reagents with reference to the immunohistochemical staining procedure can be found in Table 1 . Table 1. Details of ancillary reagents for immunohistochemistry. Process Reagent Manufacturer Catalogue Number Concentration Fixation Neutral Buffered Formaldehyde Sigma HT501128 10% Pretreatment ER1 (Sodium Citrate, pH6) Leica Biosystems AR9961 Proprietary ER2 (Tris/EDTA, pH9) Leica Biosystems AR9640 Proprietary Enzyme 1 (Proteinase K) Leica Biosystems AR9551 100 µg/ml Staining Peroxide Block Leica Biosystems DS9263 Proprietary Streptavidin – HRP Leica Biosystems DS9263 Proprietary Diaminobenizidine (DAB) Leica Biosystems DS9263 Proprietary Haematoxylin Leica Biosystems DS9263 Proprietary DAB Enhancer Leica Biosystems AR9452 Proprietary Washes/Blocks Bond Wash (Tris Buffer) Leica Biosystems AR9590 Proprietary Antibody Diluent Leica Biosystems AR9352 Proprietary Avidin/Biotin Block Vector Laboratories SP-2001 Proprietary Antibody details Anti-ARID1A is a monospecific rabbit polyclonal generated to a PrEST sequence – PGLGNVAMGPRQHYPYGGPYDRVRTEPGIGPEGNMSTGAPQPNLMPSNPDSGMYSPSRYPPQQQQQQQQRHDSYGNQFSTQGTPSGSPFPSQQTTMYQQQQQNYK ( Table 2 ). The homology of the PrEST sequence used as immunogen is 95%, when verified with BLAST against the mouse sequence. The lot number used for all validations was A40072 and for subsequent staining was D81856. A concentration of 1 µg/ml was used for initial validations and 0.5 µg/ml for final runs. Table 2. Details of primary and secondary antibodies. Antibody Manufacturer Catalogue Number RRID ARID1A Atlas Antibodies HPA005456 AB_1078205 GAPDH (Clone 14C10) Cell Signaling 2118 AB_561053 Donkey anti-Rabbit Biotin Jackson Immunoresearch 711-065-152 AB_2333077 Goat anti-Rabbit IRDye 680LT Li-Cor Biosciences 926-68021 AB_10706309 Goat anti-Rabbit IRDye 800CW Li-Cor Biosciences 926-32213 AB_621848 Donkey anti-rabbit biotin (Jackson Immunoresearch, Table 2 ) is specific for Rabbit IgG (Heavy and Light chains) and was affinity purified to remove cross-reactions to Bovine, Chicken, Goat, Guinea Pig, Syrian Hamster, Horse, Human, Mouse, Rat and Sheep. All slides were stained with a concentration of 4.8 µg/ml. Anti-GAPDH was used as a loading control for Western blots and was a rabbit monoclonal (Cell Signaling, Table 2 ). Detection antibody for the Western blot for ARID1a was Goat anti-rabbit IR Dye 680LT (Li-Cor Biosciences, Table 2 ) used at a concentration of 0.1 µg/ml and detection of GAPDH was Goat anti-rabbit IR Dye 800CW (Li-Cor Biosciences, Table 2 ). Tissue details All tissues and cell pellets used during the validation were fixed in Neutral Buffered Formaldehyde as specified ( Table 3 ) before being transferred directly to 70% ethanol for no longer than 3 days. Tissue processing was conducted on a Leica ASP300 through graded ethanols before clearing in xylene and impregnation in molten paraffin wax (Fisher). All tissue sections were cut on a Leica rotary microtome at 3 µm. Arid 1a -/- mice were created by crossing Floxed Arid1a mice with ROSA26 Cre-ERT2 mice and resultant genotyping. Loss of Arid1a expression is expected following intraperitoneal injection of Tamoxifen. Floxed Arid1a mice were a gift from Dr. Peri Tate, Sanger Institute, Hinxton UK; ROSA26 Cre-ERT2 mice were a gift from Prof Chambon, IGBMC, France. ES-2 cells were purchased from ATCC and RMG-II were a gift from Prof Huntsman, British Columbia Cancer Agency, Vancouver, Canada. Table 3. Details of tissue and cell pellet used during the validation. Species Tissue Type Strain/Cell line Details Fixation Time Murine Uterus C57Bl6 Female 16 hrs Murine Uterus C57Bl6 KO Arid1a fl/fl Female 24 hrs Human Cell Pellet ES-2 20 hrs Human Cell Pellet RMG-II 20 hrs Experiment details Western blot. Protein was extracted from the two clear cell carcinoma cell lines using a Tris-EDTA lysis buffer and run on a non-denaturing 3–8% Tris-acetate gel (Life Technologies). Following electrophoresis, the transfer membrane was probed with 0.2 µg/ml of anti-rabbit ARID1A (HPA005456) at 4°C overnight and 0.1 µg/ml anti-GAPDH (14C10) for the same length of time. Detection of the anti-rabbit ARID1A was with Goat anti-rabbit IRDye 680LT (Li-Cor Biosciences) and the GAPDH was with Goat anti-rabbit IRDye 800CW (Li-Cor Biosciences) both at 0.1 µg/ml. Immunohistochemistry. Slides were deparaffinised and rehydrated on a Leica ST5020 using Xylene (Sigma) for 2 × 10 mins and ethanol (Fisher), 2 × 100% ethanol followed by 1 × 70% ethanol for 5 mins each. Following staining, all slides were dehydrated, cleared and mounted and coverslipped in DPX (Fisher). The antibody was validated on a Leica BondMax instrument using a Leica Intense R kit to a standardised in-house protocol. All reagents were from Leica as part of the Intense R kit and were conducted at room temperature, unless otherwise specified. All staining steps included individual washes in Leica Bond Wash after each step, as part of the protocol ( Table 4 ). A full protocol for the validated conditions can be found in the supplementary material . In this protocol, the step named “primary” refers to the anti-ARID1a primary antibody. Table 4. Staining protocol for ARID1A immunohistochemistry. Protocol steps Reagent Time (mins) Antigen Retrieval ER1 20 Or ER2 20 Or Enz1 10 Staining Peroxide Block 5 Avidin 10 Biotin 10 ARID1A 15 Donkey anti-rabbit Biotin 8 SA-HRP 8 DAB 5 DAB Enhancer 10 Counterstaining Haematoxylin 5 A slide using the same conditions and retrieval but omitting the primary antibody was used to control for any background staining due to the retrieval and detection steps. Imaging. All slides were digitised using a Leica Scanscope AT2 at 0.5 µm/pixel resolution. Datasets can be viewed by downloading the Leica Imagescope free viewer at http://www.leicabiosystems.com/pathology-imaging/aperio-epathology/integrate/imagescope/ . Results To determine the correct cell line to utilise and to confirm the equivocal Western blot data from Human Protein Atlas, the antibody was used to stain a Western blot of two cell lines; ES-2 and RMG-II, both of which are cell lines derived from clear cell carcinoma and have been previously demonstrated as ARID1a wild-type and mutated, respectively 9 . It could be demonstrated that the HPA ARID1A antibody showed positive expression in ES-2 cell lines at the expected size of 270 kDa and no staining for RMG-II. The loading control of GAPDH showed that there were no loading issues ( Figure 1 ). Thus, these cell lines were chosen to be grown, formalin fixed and processed into paraffin wax for immunohistochemical validation. Figure 1. Western blot of ES2 and RMG-II clear cell carcinoma cells lines. ARID1A ( red band ) can be seen to be present at approximately 270kD in ES2 cell line only. GAPDH at 37kD ( green band ) represents loading control. For immunohistochemical validation, ES-2 and RMG-II cell lines were stained using three antigen retrieval conditions; ER1 (Sodium Citrate, pH6), ER2 (Tris/EDTA, pH9) and Enzyme 1 (Proteinase K, 100 µg/ml) at a fixed antibody concentration of 1 µg/ml. The enzyme retrieval demonstrated no nuclear signal for either ES2 or RMG-II cell pellets and was discarded for future work ( Figure 2 ; Dataset a ). The ER2 condition did demonstrate significant nuclear staining in the ES2 cell pellet with minimal background staining in the RMG-II cell pellet ( Figure 2 ; Dataset b ). However, the staining in the ER1 condition was determined to give the best signal:noise ratio with no background cytoplasmic staining and crisp nuclear staining for the cell pellet ( Figure 2 ; Dataset c ). Control slides, omitting the primary antibody, were negative except for the ER2 condition in the RMG-II cell pellet where a weak cytoplasmic background could be seen ( Figure 2 ; Dataset d ). Thus there was minimal background inherent in the staining procedure. It was therefore determined that the antibody showed specificity for formalin-fixed paraffin embedded tissues and could be run on murine tissue. Figure 2. ES2 and RMG-II cell lines stained by immunohistochemistry with anti-ARID1A using three antigen retrieval conditions, ER1, ER2 and Enzyme 1. NPA denotes No Primary antibody control and represents the ER2 condition. Bar = 200 µm. Murine uterine tissue was used as positive control tissue samples, given the literature data on cell lines and endometrial tissue. The ER1 condition at 1 µg/ml demonstrated clean nuclear staining in the uterine epithelial compartment as well as nuclear staining of stromal cells. However, the nuclear staining in the stroma was not universal and distinct negative nuclei could be seen ( Figure 3A ; Dataset e ). There was no cytoplasmic or extracellular stromal background staining present and the antibody titrated successfully losing the intensity of staining, as expected ( Dataset e ). Following this, a concentration of 0.5 µg/ml was used for future preparations which provided clear and consistent staining in repeated batches using a different antibody lot ( Dataset f ). A No Primary antibody control (NPA) showed no staining in the epithelial or nuclear compartment ( Figure 3B ; Dataset e ). Figure 3. A ) Murine uterine tissue stained by immunohistochemistry with anti-ARID1A using the ER1 condition and a concentration of 1 ug/ml demonstrating clear nuclear staining of the epithelial compartment ( E ) and negative nuclei in the stromal compartment ( S ). B ) No primary antibody control of a similar area of epithelium/stroma. Bar = 100 µm. Finally, when applied to a genetically engineered, tamoxifen-induced, Arid1a knockout mouse model, the staining in the uterine epithelium could be almost completely abrogated (Arrow, Figure 4b ) when compared to the same area in a wild-type animal (Arrow, Figure 4a ) with a small focal area of epithelial staining still present. There was no effect of the KO on the staining in the stromal compartment. Figure 4. Murine uterine tissue stained by immunohistochemistry with anti-ARID1A demonstrating nuclear staining in wild-type mice ( A ) but loss of epithelial staining after ARID1A knock-out in Arid1a fl/fl mice ( B ). Bar = 100 μm. Dataset 1. Whole slide images from antibody validation of HPA005456 for immunohistochemistry - Version 2. Detailed legends for each dataset (Datasets a–f) can be found in the text file provided. For Version 2 an additional image was added to Dataset e. Conclusions It is clear from the use of ES2 and RMG-II cell lines that the Atlas Antibodies ARID1A antibody is specific for ARID1A in both Western blots and formalin-fixed paraffin embedded preparations of human origin and, coupled with the literature evidence, that it is validated in human tissue. The staining pattern when applied to murine uterus showing a clear nuclear pattern, where there is a high level of sequence homology between the two species, is again consistent with the literature on this protein. When stained on an ARID1a knockout mouse model, the staining could be almost completely abrogated in the epithelial compartment but not in the stroma. Knockout mice generated in this manner are almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (Tamoxifen) penetration or failure of the ligand to induce recombination, thus explaining the small focal area of epithelial staining. The difference in staining in the two compartments is also likely related to the same effect, as all other controls, such as omission of primary antibody remained negative. Thus, given the overwhelming data from other sources, it is likely the stromal staining reflects continuing Arid1a expression in this specific model system. Therefore, in conclusion when taken in combination, it is clear that the anti-human ARID1a antibody is cross-reactive with murine tissue and can be used for this purpose. Data availability F1000Research: Dataset 1. Whole slide images from antibody validation of HPA005456 for immunohistochemistry - Version 2, 10.5256/f1000research.5514.d41579 10 Author contributions WH wrote and conceived the idea behind the article, IG performed the Western blots and mouse experiments and requested validation of ARID1A in murine tissue, JM performed all immunohistochemical staining. Competing interests No competing interests were disclosed. Grant information WH and JM are funded by Cancer Research UK, IG is funded by an MRC Clinical Research Training Fellowship, grant number G1001957. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Acknowledgements The authors would like to acknowledge the University of Cambridge, Cancer Research UK and Hutchison Whampoa Ltd. Dr. Peri Tate, Wellcome Trust Sanger Institute, Prof Chambon, IGBMC and Prof Huntsman, British Columbia Cancer Agency for their kind gifts of materials and the staff at the Histopathology/ISH core facility at the Cancer Research UK Cambridge Institute for their assistance in preparing materials for this publication. Faculty Opinions recommended References 1. Streppel MM, Lata S, DelaBastide M, et al. : Next-generation sequencing of endoscopic biopsies identifies ARID1A as a tumor-suppressor gene in Barrett's esophagus. Oncogene. 2014; 33 (3): 347–357. PubMed Abstract | Publisher Full Text | Free Full Text 2. Wiegand KC, Hennessy BT, Leung S, et al. : A functional proteogenomic analysis of endometrioid and clear cell carcinomas using reverse phase protein array and mutation analysis: protein expression is histotype-specific and loss of ARID1A /BAF250a is associated with AKT phosphorylation. BMC Cancer. 2014; 14 : 120. PubMed Abstract | Publisher Full Text | Free Full Text 3. Wiegand KC, Shah SP, Al-Agha OM, et al. : ARID1A mutations in endometriosis-associated ovarian carcinomas. N Engl J Med. 2010; 363 (16): 1532–1543. PubMed Abstract | Publisher Full Text | Free Full Text 4. Dulak AM, Stojanov P, Peng S, et al. : Exome and whole-genome sequencing of esophageal adenocarcinoma identifies recurrent driver events and mutational complexity. Nat Genet. 2013; 45 (5): 478–486. PubMed Abstract | Publisher Full Text | Free Full Text 5. Nilsson P, Paavilainen L, Larsson K, et al. : Towards a human proteome atlas: high-throughput generation of mono-specific antibodies for tissue profiling. Proteomics. 2005; 5 (17): 4327–4337. PubMed Abstract | Publisher Full Text 6. Human Protein Atlas. Reference Source 7. Wang K, Kan J, Yuen ST, et al. : Exome sequencing identifies frequent mutation of ARID1A in molecular subtypes of gastric cancer. Nat Genet. 2011; 43 (12): 1219–1223. PubMed Abstract | Publisher Full Text 8. Yamamoto S, Tsuda H, Takano M, et al. : Loss of ARID1A protein expression occurs as an early event in ovarian clear-cell carcinoma development and frequently coexists with PIK3CA mutations. Mod Pathol. 2012; 25 (4): 615–624. PubMed Abstract | Publisher Full Text 9. Anglesio MS, Wiegand KC, Melnyk N, et al. : Type-specific cell line models for type-specific ovarian cancer research. PLoS One. 2013; 8 (9): e72162. PubMed Abstract | Publisher Full Text | Free Full Text 10. Howat WJ, Miller JL, Gounaris I: Whole slide images from antibody validation of HPA005456 for immunohistochemistry - Version 2. F1000Research. 2014. Data Source Supplementary material Comments on this article Comments (1) Version 3 VERSION 3 PUBLISHED 04 Jun 2015 Revised Comment ADD YOUR COMMENT Version 2 VERSION 2 PUBLISHED 06 Jan 2015 Revised Discussion is closed on this version, please comment on the latest version above. Reader Comment 12 Jan 2015 Jan Voskuil , Everest Biotech Ltd, Oxfordshire, UK 12 Jan 2015 Reader Comment Well done! Competing Interests: No competing interests were disclosed. Well done! Well done! Competing Interests: No competing interests were disclosed. Close Report a concern Discussion is closed on this version, please comment on the latest version above. Author details Author details 1 Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK Competing interests No competing interests were disclosed. Grant information WH and JM are funded by Cancer Research UK, IG is funded by an MRC Clinical Research Training Fellowship, grant number G1001957. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Article Versions (3) version 3 Revised Published: 04 Jun 2015, 3:244 https://doi.org/10.12688/f1000research.5514.3 version 2 Revised Published: 06 Jan 2015, 3:244 https://doi.org/10.12688/f1000research.5514.2 version 1 Published: 15 Oct 2014, 3:244 https://doi.org/10.12688/f1000research.5514.1 Copyright © 2015 Howat W et al . This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Data associated with the article are available under the terms of the Creative Commons Zero "No rights reserved" data waiver (CC0 1.0 Public domain dedication). Download Export To Sciwheel Bibtex EndNote ProCite Ref. Manager (RIS) Sente metrics Views Downloads F1000Research - - PubMed Central info_outline Data from PMC are received and updated monthly. - - Citations open_in_new 0 open_in_new 0 open_in_new SEE MORE DETAILS CITE how to cite this article Howat W, Miller J and Gounaris I. Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.12688/f1000research.5514.3 ) NOTE: If applicable, it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS track receive updates on this article Track an article to receive email alerts on any updates to this article. TRACK THIS ARTICLE Share Open Peer Review Current Reviewer Status: ? Key to Reviewer Statuses VIEW HIDE Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Version 2 VERSION 2 PUBLISHED 06 Jan 2015 Revised Views 0 Cite How to cite this report: McQuaid S. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7236 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7236 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 12 Jan 2015 Stephen McQuaid , Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.6423.r7236 All concerns ... Continue reading READ ALL All concerns have been addressed. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT McQuaid S. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7236 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7236 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Chalmers AD. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7235 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7235 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 09 Jan 2015 Andrew D. Chalmers , Department of Biology and Biochemistry, University of Bath, Bath, UK Approved VIEWS 0 https://doi.org/10.5256/f1000research.6423.r7235 The authors have addressed my comments/concerns satisfactorily ... Continue reading READ ALL The authors have addressed my comments/concerns satisfactorily and I am happy to approve the manuscript. Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Chalmers AD. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7235 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7235 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Respond or Comment COMMENT ON THIS REPORT Version 1 VERSION 1 PUBLISHED 15 Oct 2014 Views 0 Cite How to cite this report: McQuaid S. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6848 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6848 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 02 Dec 2014 Stephen McQuaid , Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.5886.r6848 This article, by Howatt et al. validates an antibody to ARID1A in murine FFPE samples, is a good example of the correct validations procedures that must be met when validating an antibody for use in tissue sections. Issues are relatively ... Continue reading READ ALL This article, by Howatt et al. validates an antibody to ARID1A in murine FFPE samples, is a good example of the correct validations procedures that must be met when validating an antibody for use in tissue sections. Issues are relatively minor: What was the rationale for a starting concentration of 1μg/ml for initial validations? There is a focal region of the KO epithelium which clearly appears to be positive. The authors would need to explain this. Can the authors please add more discussion of the nature of the staining of the stromal cells. Do they consider this to be non-specific? Is such staining of stroma present in human tissue sections? Control slides, omitting primary antibody, were carried out during the cell line phase of the experiments. Were such slides run on the murine tissue sections and can the results be included in the data. I note that the stromal staining was still present at selected concentration of 0.5μg/ml (dataset f) Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT McQuaid S. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6848 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6848 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 06 Jan 2015 Will Howat , Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK 06 Jan 2015 Author Response Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of ... Continue reading Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of 1:100 from the manufacturers provided stock concentration and we perform 3 retrieval methods; Sodium Citrate pH6, Tris EDTA pH9 & Proteinase K. We titrate as appropriate following establishment of the optimal retrieval method. The manufacturers original stock concentration was 100ug/ml, hence the 1ug/ml starting concentration. They have subsequently changed their stock concentration to 200ug/ml. We believe that the knockout model recombining a floxed Arid1a with CRE-ERT2 is not 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Thus explaining the area of focal staining in the epithelium. Similarly, although we have not investigated this further, we believe that the same problem underlies the stromal staining and is likely due to penetrance of the tamoxifen after being delivered intraperitoneally. Thus, given the wealth of data and the no primary antibody controls, now included in Fig 3, we feel that the stromal staining is not non-specific. Stromal staining does indeed occur in human tissue. All of the above have now been included in the conclusion section of the article. Kind Regards, Will Howat Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of 1:100 from the manufacturers provided stock concentration and we perform 3 retrieval methods; Sodium Citrate pH6, Tris EDTA pH9 & Proteinase K. We titrate as appropriate following establishment of the optimal retrieval method. The manufacturers original stock concentration was 100ug/ml, hence the 1ug/ml starting concentration. They have subsequently changed their stock concentration to 200ug/ml. We believe that the knockout model recombining a floxed Arid1a with CRE-ERT2 is not 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Thus explaining the area of focal staining in the epithelium. Similarly, although we have not investigated this further, we believe that the same problem underlies the stromal staining and is likely due to penetrance of the tamoxifen after being delivered intraperitoneally. Thus, given the wealth of data and the no primary antibody controls, now included in Fig 3, we feel that the stromal staining is not non-specific. Stromal staining does indeed occur in human tissue. All of the above have now been included in the conclusion section of the article. Kind Regards, Will Howat Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 06 Jan 2015 Will Howat , Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK 06 Jan 2015 Author Response Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of ... Continue reading Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of 1:100 from the manufacturers provided stock concentration and we perform 3 retrieval methods; Sodium Citrate pH6, Tris EDTA pH9 & Proteinase K. We titrate as appropriate following establishment of the optimal retrieval method. The manufacturers original stock concentration was 100ug/ml, hence the 1ug/ml starting concentration. They have subsequently changed their stock concentration to 200ug/ml. We believe that the knockout model recombining a floxed Arid1a with CRE-ERT2 is not 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Thus explaining the area of focal staining in the epithelium. Similarly, although we have not investigated this further, we believe that the same problem underlies the stromal staining and is likely due to penetrance of the tamoxifen after being delivered intraperitoneally. Thus, given the wealth of data and the no primary antibody controls, now included in Fig 3, we feel that the stromal staining is not non-specific. Stromal staining does indeed occur in human tissue. All of the above have now been included in the conclusion section of the article. Kind Regards, Will Howat Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of 1:100 from the manufacturers provided stock concentration and we perform 3 retrieval methods; Sodium Citrate pH6, Tris EDTA pH9 & Proteinase K. We titrate as appropriate following establishment of the optimal retrieval method. The manufacturers original stock concentration was 100ug/ml, hence the 1ug/ml starting concentration. They have subsequently changed their stock concentration to 200ug/ml. We believe that the knockout model recombining a floxed Arid1a with CRE-ERT2 is not 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Thus explaining the area of focal staining in the epithelium. Similarly, although we have not investigated this further, we believe that the same problem underlies the stromal staining and is likely due to penetrance of the tamoxifen after being delivered intraperitoneally. Thus, given the wealth of data and the no primary antibody controls, now included in Fig 3, we feel that the stromal staining is not non-specific. Stromal staining does indeed occur in human tissue. All of the above have now been included in the conclusion section of the article. Kind Regards, Will Howat Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Views 0 Cite How to cite this report: Chalmers AD. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6651 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6651 NOTE: it is important to ensure the information in square brackets after the title is included in this citation. Close Copy Citation Details Reviewer Report 21 Nov 2014 Andrew D. Chalmers , Department of Biology and Biochemistry, University of Bath, Bath, UK Approved with Reservations VIEWS 0 https://doi.org/10.5256/f1000research.5886.r6651 The manuscript by Will Howat and colleagues validates an anti-ARID1A antibody and provides a good example of an antibody validation paper. In particular the use of negative cell lines and knockout mouse tissue demonstrate the extent of specificity of the ... Continue reading READ ALL The manuscript by Will Howat and colleagues validates an anti-ARID1A antibody and provides a good example of an antibody validation paper. In particular the use of negative cell lines and knockout mouse tissue demonstrate the extent of specificity of the antibody. The data is mostly well presented and clearly explained in the text. The manuscript would be suitable for indexing if some, mostly minor, issues can be addressed. I think the title should mention “antibody” to make clear it’s an antibody that is being validated. The authors say there is 95% sequence homology between human and mouse, it would be interesting to see how conserved the sequence the antibody was raised against is. The Materials and Methods give a good overview of the reagents and methods used for the IHC, but there is little information about the western blotting. The reagents and methods used should be added. It would be good to cite the source of the cells used and the mouse tissue. I was not clear if the lack of staining in RMG-II cells was expected or an unexpected but useful result? This should be explained. Figure 1 legend. Should read “represents the loading control” In figure 4B the KO epithelium is clearly negative, except one region in the top left which looks positive, I wondered what the authors felt about this? It is interesting that the stromal staining appears to be non-specific, I wonder if the authors ever did a no primary control? Does the stroma still stain? Also it would be worth mentioning this staining in the conclusions. Author contributions. Should it read “conceived the idea behind the article” or similar wording ? Competing Interests: No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. Close READ LESS CITE CITE HOW TO CITE THIS REPORT Chalmers AD. Reviewer Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6651 ) The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6651 NOTE: it is important to ensure the information in square brackets after the title is included in all citations of this article. COPY CITATION DETAILS Report a concern Author Response 06 Jan 2015 Will Howat , Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK 06 Jan 2015 Author Response Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched ... Continue reading Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched the protein sequence and it confirms 95% homology for the sequence used for immunisations. This has been added to the methods. We accept that the methods detailing the Western blotting are more limited than the IHC methods, as is the case of the methods detailing the production of the knockout mouse model. However, we feel that the article was designed as an example of antibody validation for immunohistochemistry and that while western blotting provides important additional data, it is not the focus of the article. We thus feel that there is sufficient information in the details behind the western blotting to repeat the experiment, without clouding the article with the full methods. The sources of the cells and mouse tissue have now been cited. The lack of staining of RMG-II cells was consistent with the literature (Anglesio et al. ) and this information has been added to the results section. The figure legend has been modified. We believe that the focal staining is a result of incomplete recombination floxed Arid1a allele and Cre/ERT2. The resulting knockout is almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Similarly, we believe that the staining in the stromal compartment is specific but represents a failure of delivery of Tamoxifen or of recombination. The data from no primary antibody (NPA) control which are all completely clean, now included in Fig 3, helps to demonstrate this. We have included these points in the conclusion. The author contributions have been modified. Kind Regards, Will Howat Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched the protein sequence and it confirms 95% homology for the sequence used for immunisations. This has been added to the methods. We accept that the methods detailing the Western blotting are more limited than the IHC methods, as is the case of the methods detailing the production of the knockout mouse model. However, we feel that the article was designed as an example of antibody validation for immunohistochemistry and that while western blotting provides important additional data, it is not the focus of the article. We thus feel that there is sufficient information in the details behind the western blotting to repeat the experiment, without clouding the article with the full methods. The sources of the cells and mouse tissue have now been cited. The lack of staining of RMG-II cells was consistent with the literature (Anglesio et al. ) and this information has been added to the results section. The figure legend has been modified. We believe that the focal staining is a result of incomplete recombination floxed Arid1a allele and Cre/ERT2. The resulting knockout is almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Similarly, we believe that the staining in the stromal compartment is specific but represents a failure of delivery of Tamoxifen or of recombination. The data from no primary antibody (NPA) control which are all completely clean, now included in Fig 3, helps to demonstrate this. We have included these points in the conclusion. The author contributions have been modified. Kind Regards, Will Howat Competing Interests: No competing interests were disclosed. Close Report a concern Respond or Comment COMMENTS ON THIS REPORT Author Response 06 Jan 2015 Will Howat , Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK 06 Jan 2015 Author Response Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched ... Continue reading Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched the protein sequence and it confirms 95% homology for the sequence used for immunisations. This has been added to the methods. We accept that the methods detailing the Western blotting are more limited than the IHC methods, as is the case of the methods detailing the production of the knockout mouse model. However, we feel that the article was designed as an example of antibody validation for immunohistochemistry and that while western blotting provides important additional data, it is not the focus of the article. We thus feel that there is sufficient information in the details behind the western blotting to repeat the experiment, without clouding the article with the full methods. The sources of the cells and mouse tissue have now been cited. The lack of staining of RMG-II cells was consistent with the literature (Anglesio et al. ) and this information has been added to the results section. The figure legend has been modified. We believe that the focal staining is a result of incomplete recombination floxed Arid1a allele and Cre/ERT2. The resulting knockout is almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Similarly, we believe that the staining in the stromal compartment is specific but represents a failure of delivery of Tamoxifen or of recombination. The data from no primary antibody (NPA) control which are all completely clean, now included in Fig 3, helps to demonstrate this. We have included these points in the conclusion. The author contributions have been modified. Kind Regards, Will Howat Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched the protein sequence and it confirms 95% homology for the sequence used for immunisations. This has been added to the methods. We accept that the methods detailing the Western blotting are more limited than the IHC methods, as is the case of the methods detailing the production of the knockout mouse model. However, we feel that the article was designed as an example of antibody validation for immunohistochemistry and that while western blotting provides important additional data, it is not the focus of the article. We thus feel that there is sufficient information in the details behind the western blotting to repeat the experiment, without clouding the article with the full methods. The sources of the cells and mouse tissue have now been cited. The lack of staining of RMG-II cells was consistent with the literature (Anglesio et al. ) and this information has been added to the results section. The figure legend has been modified. We believe that the focal staining is a result of incomplete recombination floxed Arid1a allele and Cre/ERT2. The resulting knockout is almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Similarly, we believe that the staining in the stromal compartment is specific but represents a failure of delivery of Tamoxifen or of recombination. The data from no primary antibody (NPA) control which are all completely clean, now included in Fig 3, helps to demonstrate this. We have included these points in the conclusion. The author contributions have been modified. Kind Regards, Will Howat Competing Interests: No competing interests were disclosed. Close Report a concern COMMENT ON THIS REPORT Comments on this article Comments (1) Version 3 VERSION 3 PUBLISHED 04 Jun 2015 Revised Comment ADD YOUR COMMENT Version 2 VERSION 2 PUBLISHED 06 Jan 2015 Revised Discussion is closed on this version, please comment on the latest version above. Reader Comment 12 Jan 2015 Jan Voskuil , Everest Biotech Ltd, Oxfordshire, UK 12 Jan 2015 Reader Comment Well done! Competing Interests: No competing interests were disclosed. Well done! Well done! Competing Interests: No competing interests were disclosed. Close Report a concern Discussion is closed on this version, please comment on the latest version above. keyboard_arrow_left keyboard_arrow_right Open Peer Review Reviewer Status info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions Reviewer Reports Invited Reviewers 1 2 Version 3 (revision) 04 Jun 15 Version 2 (revision) 06 Jan 15 read read Version 1 15 Oct 14 read read Andrew D. Chalmers , University of Bath, Bath, UK Stephen McQuaid , Queen's University Belfast, Belfast, UK Comments on this article All Comments (1) Add a comment Sign up for content alerts Sign Up You are now signed up to receive this alert Browse by related subjects keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2015 McQuaid S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 12 Jan 2015 | for Version 2 Stephen McQuaid , Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK 0 Views copyright © 2015 McQuaid S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions All concerns have been addressed. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) McQuaid S. Peer Review Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7236) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7236 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2015 Chalmers A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 09 Jan 2015 | for Version 2 Andrew D. Chalmers , Department of Biology and Biochemistry, University of Bath, Bath, UK 0 Views copyright © 2015 Chalmers A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (0) Approved info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The authors have addressed my comments/concerns satisfactorily and I am happy to approve the manuscript. Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. reply Respond to this report Responses (0) Chalmers AD. Peer Review Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.6423.r7235) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/3-244/v2#referee-response-7235 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2014 McQuaid S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 02 Dec 2014 | for Version 1 Stephen McQuaid , Center for Cancer Research and Cell Biology, Queen's University Belfast, Belfast, UK 0 Views copyright © 2014 McQuaid S. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions This article, by Howatt et al. validates an antibody to ARID1A in murine FFPE samples, is a good example of the correct validations procedures that must be met when validating an antibody for use in tissue sections. Issues are relatively minor: What was the rationale for a starting concentration of 1μg/ml for initial validations? There is a focal region of the KO epithelium which clearly appears to be positive. The authors would need to explain this. Can the authors please add more discussion of the nature of the staining of the stromal cells. Do they consider this to be non-specific? Is such staining of stroma present in human tissue sections? Control slides, omitting primary antibody, were carried out during the cell line phase of the experiments. Were such slides run on the murine tissue sections and can the results be included in the data. I note that the stromal staining was still present at selected concentration of 0.5μg/ml (dataset f) Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 06 Jan 2015 Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK Dear Dr McQuaid, Thank you for your comments which we agree with and have taken on board. Specifically: All of the validations conducted at the Histopathology/ISH facility start with a dilution of 1:100 from the manufacturers provided stock concentration and we perform 3 retrieval methods; Sodium Citrate pH6, Tris EDTA pH9 & Proteinase K. We titrate as appropriate following establishment of the optimal retrieval method. The manufacturers original stock concentration was 100ug/ml, hence the 1ug/ml starting concentration. They have subsequently changed their stock concentration to 200ug/ml. We believe that the knockout model recombining a floxed Arid1a with CRE-ERT2 is not 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Thus explaining the area of focal staining in the epithelium. Similarly, although we have not investigated this further, we believe that the same problem underlies the stromal staining and is likely due to penetrance of the tamoxifen after being delivered intraperitoneally. Thus, given the wealth of data and the no primary antibody controls, now included in Fig 3, we feel that the stromal staining is not non-specific. Stromal staining does indeed occur in human tissue. All of the above have now been included in the conclusion section of the article. Kind Regards, Will Howat View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern McQuaid S. Peer Review Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6848) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6848 keyboard_arrow_left Back to all reports Reviewer Report 0 Views copyright © 2014 Chalmers A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 21 Nov 2014 | for Version 1 Andrew D. Chalmers , Department of Biology and Biochemistry, University of Bath, Bath, UK 0 Views copyright © 2014 Chalmers A. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. format_quote Cite this report speaker_notes Responses (1) Approved With Reservations info_outline Alongside their report, reviewers assign a status to the article: Approved The paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved Fundamental flaws in the paper seriously undermine the findings and conclusions The manuscript by Will Howat and colleagues validates an anti-ARID1A antibody and provides a good example of an antibody validation paper. In particular the use of negative cell lines and knockout mouse tissue demonstrate the extent of specificity of the antibody. The data is mostly well presented and clearly explained in the text. The manuscript would be suitable for indexing if some, mostly minor, issues can be addressed. I think the title should mention “antibody” to make clear it’s an antibody that is being validated. The authors say there is 95% sequence homology between human and mouse, it would be interesting to see how conserved the sequence the antibody was raised against is. The Materials and Methods give a good overview of the reagents and methods used for the IHC, but there is little information about the western blotting. The reagents and methods used should be added. It would be good to cite the source of the cells used and the mouse tissue. I was not clear if the lack of staining in RMG-II cells was expected or an unexpected but useful result? This should be explained. Figure 1 legend. Should read “represents the loading control” In figure 4B the KO epithelium is clearly negative, except one region in the top left which looks positive, I wondered what the authors felt about this? It is interesting that the stromal staining appears to be non-specific, I wonder if the authors ever did a no primary control? Does the stroma still stain? Also it would be worth mentioning this staining in the conclusions. Author contributions. Should it read “conceived the idea behind the article” or similar wording ? Competing Interests No competing interests were disclosed. I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above. reply Respond to this report Responses (1) Author Response 06 Jan 2015 Will Howat, Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge, CB2 0RE, UK Dear Dr Chalmers, Many thanks for your comments which we agree with and have taken on board. Specifically: We have modified the title to read "Application of anti-ARID1a antibody..." We have BLAST searched the protein sequence and it confirms 95% homology for the sequence used for immunisations. This has been added to the methods. We accept that the methods detailing the Western blotting are more limited than the IHC methods, as is the case of the methods detailing the production of the knockout mouse model. However, we feel that the article was designed as an example of antibody validation for immunohistochemistry and that while western blotting provides important additional data, it is not the focus of the article. We thus feel that there is sufficient information in the details behind the western blotting to repeat the experiment, without clouding the article with the full methods. The sources of the cells and mouse tissue have now been cited. The lack of staining of RMG-II cells was consistent with the literature (Anglesio et al. ) and this information has been added to the results section. The figure legend has been modified. We believe that the focal staining is a result of incomplete recombination floxed Arid1a allele and Cre/ERT2. The resulting knockout is almost never 100% complete as in some cells recombination will not be induced due to issues such as low ligand (tamoxifen) penetration or failure of the ligand to induce recombination. Similarly, we believe that the staining in the stromal compartment is specific but represents a failure of delivery of Tamoxifen or of recombination. The data from no primary antibody (NPA) control which are all completely clean, now included in Fig 3, helps to demonstrate this. We have included these points in the conclusion. The author contributions have been modified. Kind Regards, Will Howat View more View less Competing Interests No competing interests were disclosed. reply Respond Report a concern Chalmers AD. Peer Review Report For: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry [version 3; peer review: 2 approved] . F1000Research 2015, 3 :244 ( https://doi.org/10.5256/f1000research.5886.r6651) NOTE: it is important to ensure the information in square brackets after the title is included in this citation. The direct URL for this report is: https://f1000research.com/articles/3-244/v1#referee-response-6651 Alongside their report, reviewers assign a status to the article: Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit. Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions Click here to access the data. The problem Spreadsheet data files may not format correctly if your computer is using different default delimiters (symbols used to separate values into separate cells) - a spreadsheet created in one region is sometimes misinterpreted by computers in other regions. You can change the regional settings on your computer so that the spreadsheet can be interpreted correctly. How to fix it Save downloaded CSV file Open spreadsheet program (e.g. Excel) Click the ‘Data’ tab at the top Click the ‘From text’ icon (top left) Browse for downloaded CSV file, click ‘Import’ Ensure ‘Delimited’ radio button is selected, click ‘Next’ Check one of the appropriate delimiter checkboxes (you can visualize the formatting by looking at the data preview below these options) Click ‘Finish’ Downloaded data do not display as expected? Download the data Dataset citation: Howat W, Miller J and Gounaris I. Dataset 1 in: Application of ARID1A to murine formalin-fixed paraffin embedded tissue using immunohistochemistry. 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