Cytochemical localization of ZIO-stained compartments in Trypanosoma cruzi

Cytochemical localization of ZIO-stained compartments in Trypanosoma cruzi | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Cytochemical localization of ZIO-stained compartments in Trypanosoma cruzi Wanderley Souza, Noemia Rodrigues Alves This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9430581/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract Electron microscopy cytochemistry has contributed to a better understanding of the identification of some cytoplasmic components of eukaryotic cells, even when they are not highly specific, yet they still allow identification of cell components. Here, we describe results obtained by impregnating cells with a Zinc-Osmium Tetroxide complex to label certain organelles of Trypanosoma cruzi . Labeling of some cisternae of the Golgi complex and components of the endocytic pathway, especially the reservosomes, was observed. Labeling of ER cisternae, a reticular structure located just below the portion of the plasma membrane associated with the flagellar attachment zone (FAZ), and the contractile vacuole, was observed. Variation in the intensity of the labeling pattern is probably related to the dynamics of the cell cycle. ZIO-OsO4 staining Electron microscopy Trypanosoma cruzi Figures Figure 1 Figure 2 Figure 3 Introduction Classical cytochemical methods adapted for transmission electron microscopy have made important contributions to the identification of structures and organelles in pathogenic protozoa, allowing the localization of carbohydrates on the cell surface, the characterization of organelles such as mitochondria, peroxisomes, endocytic pathways, and the lysosomal system, and the identification of lipid inclusions, among others. In some cases, these methods allow identification of the nature of the biochemical components analyzed, as in the localization of carbohydrates or enzymes. In other cases, the methods are more general in nature, only indicating whether the structures are made of lipids (as in the case of the use of osmium tetroxide/imidazole buffered solution [ 1 ] or basic proteins (as in the case of alcoholic phosphotungstic acid (PTA) [ 2 – 3 ]. Another interesting method is the use of impregnation by the Zinc-Osmium Tetroxide (ZIO) complex, initially introduced by Champy [ 4 ] and Maillet [ 5 ] in light microscopy and adapted for transmission electron microscopy [ 6 – 7 ] initially for visualization of synaptic vesicles of cholinergic junctions, without labeling other organelles located in the same place, such as mitochondria. Labeling was also found in part of the Golgi complex, especially in the GERL and lysosomes [ 8 ]. Later, Pellegrino de Iraldi and co-workers, and other authors found labeling in the endoplasmic reticulum, part of the Golgi complex, and some mitochondria [ 9 – 15 ]. Although the biochemical nature of the molecules that interact with ZIO remains unclear, there are indications of interactions with lipoprotein complexes, calcium-binding sites, and even proteins containing sulfhydryl groups [ 11 ]. More recently, Jokhura et al. [ 16 ] used ZIO impregnation for three-dimensional reconstruction of the Golgi complex in rat hepatocytes. To date, only two articles have described results obtained with ZIO in pathogenic protozoa. Benchimol and De Souza [ 17 ] showed labeling in the endoplasmic reticulum-Golgi complex, nuclear pore, and reticulum profiles located near the region of adhesion of the recurrent flagellum to the body of the protozoan Tritrichomonas foetus . Then Slomianny and Prensier [ 18 ] demonstrated labeling of the endoplasmic reticulum, Golgi complex, and lysosomal systems in Plasmodium falciparum. Although the structural organization of T. cruzi has been well established by conventional transmission electron microscopy of thin sections [Review in 19], there is little information on differential labeling of cell structures using a cytochemical approach. In this article, we describe the results obtained with ZIO for Trypanosoma cruzi , a pathogenic protozoan that causes Chagas disease, a relevant human disease, and that presents a distinct cellular organization. The results show variation in labeling of the various structures and organelles, possibly indicating heterogeneity in their composition throughout the cell cycle. Materials and Methods Obtaining the parasites T. cruzi epimastigotes were maintained in LIT culture medium supplemented with 10% of fetal bovine serum. After 4 days of inoculation, when parasite density reached 10 6 cells/ml, the parasites were collected by centrifugation. Zinc iodide-osmium technique The same protocol was applied to both cell types. Fixation was carried out in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours. Cells were rinsed in a solution of 8,5% sucrose in 0.1 M phosphate buffer, pH 7,2, 2 times for 10 minutes each. After that, the cells were rinsed with Tris aminomethane buffer (1,13M NaCl; 0,011M CaCl2; 0,03M MgCl2; 0,01M Tris aminomethane [ 20 ]. The final pH of the incubation medium was adjusted to 3.8–4.5 with 1N HCl. The cells were then incubated in ZIO solution for 18 hours, at 4°C in the dark. Shortly before the incubation, the ZIO solution was prepared as follows: 3 g Zn powder and 1 g iodine resublimed was suspended in 10 ml distilled water, separately. These solutions were mixed in a ratio of 1:1. After filtering (in a 0.45 µm Millipore filter placed in a syringe), 4 ml of this solution was mixed with 4 ml of the Tris aminomethane buffer. Immediately before using 4 parts of this solution were mixed with 1 part of a 2% OsO4 aqueous solution. After dehydration in a graded series of acetone, cells were embedded in an epoxy resin, sectioned, and observed by transmission electron microscopy. Results and Discussion In general, procedures for preparing biological specimens using metal impregnations require long incubation times. Therefore, the quality of fixation is slightly lower than that obtained by conventional methods. However, after several attempts, we established a fixation and staining procedure that proved reproducible across several experiments. Figure 1 A shows an image of the epimastigote form of T. cruzi processed in a similar way to the cells impregnated with ZIO, but without passing through this reagent. The image clearly shows the nucleus, the kinetoplast, and the Golgi complex. Figures 1 B and 1 C and Fig. 2 A-E show equivalent areas of epimastigotes impregnated with ZIO, where a clearly marked region of the Golgi complex can be seen, as well as a set of vacuoles located in the posterior region and corresponding to the endolysosomal system, consisting of the reservosomes, as well as other structures described below. Figure 1 . (A) control image of cells that were not incubated in the presence of the Zio-OsO4 solution. No labeling is seen in the various structures of the epimastigote form of T. cruzi. N, nucleus; K, kinetoplast. G, Golgi complex; (B_C) low magnification of epimastigotes incubated in the presence of Zio-OsO4. The following structures are heavily labeled: the Golgi complex region (G) and profiles of the endoplasmic reticulum (ER). The nucleus and kinetoplast are not labeled. Certainly, one of the most marked structures is the Golgi complex. However, not all the cisternae and vesicles that form this organelle were marked in the same way. In some cells, all cisternae on the cis and trans faces were marked, with the marks more pronounced in more expanded lateral regions (Fig. 2 B, E). In some cells, the label was more evident in the first cisternae (Cis face), although not in their entire extent, whereas in other cells it was evident in the last cisternae of the trans face. Images such as Fig. 2 C clearly show a non-uniform Golgi mark, with densely marked cisternae associated with dense vesicles that detach from the central cisterna. Some cells showed the electron-dense product throughout the Golgi as well as in the cisternae of the endoplasmic reticulum distributed throughout the anterior region, close to the Golgi, and in the posterior region, surrounding the reservosomes (Fig. 3 A and E). These data clearly show that the system of cisternae and vacuoles associated with the Golgi complex of T. cruzi is highly heterogeneous, indicating a dynamic activity of the glycoprotein synthesis and glycosylation system. This information is relevant because there are no detailed studies of the Golgi complex in this protozoan, although the organelle has been isolated by cell fractionation [ 21 ]. Further three-dimensional reconstruction of stained cells may add more information on this topic. The labeling on other sites deserves to be highlighted. First, a reticular system located in the lateral region of the flagellar pocket, possibly formed by cisternae and vesicles that form the contractile vacuole of T. cruzi (Fig. 1 B, 3 B). This compartment concentrates several ions and may also act as a transport route for components of the Golgi complex that are secreted into the interior of the flagellar pocket [Review in 22). Second, a reticular structure located just below the portion of the plasma membrane of the protozoan in the area close to the flagellum (Fig. 3 D). It is possible that this component is associated with the FAZ (Flagellar Attachment Zone) complex, which contains a set of important proteins such as GP 72 [ 23 ]. Third, structures are located within the endocytic system, especially in the reservosome. Based on what we know about the reservosome, it is possible that these structures correspond to the lipid component, which tends to form crystalline lipid structures [ 23 ]. Finally, we note that some epimastigotes exhibit strong nuclear membrane staining (Fig. 3 C). Declarations Author Contribution Both authros were involved in the experiments, microscopy observation and writting the article. Acknowledgements The authors thank all members of the laboratory for technical help and discussion. This work has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico-CNPq, Fundação de Apoio à Formação de Pessoal de Nível Superior-CAPES, Financiadora de Estudos e Projetos-FINEP, and Fundação Carlos Chagas Filho de Apoio à Pesquisa do Estado do Rio de Janeiro-FAPERJ References Soares MJ, de Souza MF, de Souza W (1987) Ultrastructural visualization of lipids in trypanosomatids. J Protozool. 10.1111/J.1550-7408.1987.TB03160.X Bloom FE, Aghajanian GK (1968) Fine structural and cytochemical analysis of the staining of synaptic junctions with phosphotungstic acid. J Ultrastruct Res 22:361 Souto-Padrón T, De Souza W (1979) Cytochemical analysis at the fine-structural level of trypanosomatids stained with phosphotungstic acid. J Protozool 26:551–557. 10.1111/j.1550-7408.1979.tb04194.x.PMID Champy C (1913) Granules et substances réduisant liodure d’osmium. J Anat 49:323–343 Maillet M (1962) La technique de Champy à l’osmium ioduré de potassium et la modificacion de Maillet à l’osmium-ioduré de Zinc. Trab Inst Cajal Invest Biol 54:1–36 Akert K, Moor H, Pfenninger K, Sandri C (1969) Contributions of new impregnation and freeze-etching to the problems of synaptic fine structure. In: Akert K, Waser FG (eds) Progress in Brains Research, vol 31. Elsevier Publishing Company, Amsterdm, pp 223–240 Akert K, Sandri C (1968) An electron microscopic study of zinc iodide-osmium impreganation of neurons. I. Staining of synaptic vesicles at cholinergic junctions. Brains Res 7:286–295 Marty F (1980) High voltage electron microscopy of membrane interactions in wheat. J Histochem Cytochem 28:1129–1132 Doi.0022-1554/80/10112904 $ 02.25 de Pellegrino A (1975) Electron cytochemical demonstration of -SH groups in the synaptic vesicles of photoreceptor cells with the mixture of zinc iodide-osmium tetroxide. Experientia 7:842–844. 10.1007/BF01938497 Pellegrino de Iraldi A, Cardoni R (1979) ZIO staining in synaptic vesicles of the rat pineal nerves after inhibition of serotonin and noradrenaline synthesizing enzymes. Cell Tissue Res 200:91–100. 10.1007/BF00236890 Pellegrino de Iraldi AP (1975) Localizing -SH groups in monoaminergic synaptic vesicles with the mixture of zinc iodide-osmium tetroxide (ZIO). Brain Res 94:363–367. 10.1016/0006-8993(75)90222-x Reinecke M (1981) The Zinc Iodide-Osmium Tetroxide (ZIO) method. In: Heym et al (eds) Techniques in Neuroanatomical Research. Springer-, Berlin Heidelberg, pp 292–300 Chau Y-P, Lu K-S (1994) ZIO impregnation and cytochemical localization of thiamine pyrophosphatase and acid phosphatase activities in small granule-containing (SGC) cells of rat superior cervical ganglia. Histo Histopathol 9:649–656 Schimmig B, Vicenti CA (2005) Zinc Iodide-osmium tetroxide (ZIO) reactive Golgi apparatus in the principal cells of dog epididymal epithelium. Int J Morphol 23:337–344. Doi.org/10.4067/S0717-95022005000400009 Reinecke M, Wlather C (1978) Zinc iodide-osmium tetroxide reaction: Its effect on the synaptic vesicles in locust neuromuscular junctions and its chemical basis. In: Sturgess JM (ed) Electron microscopy Vol II. The imperial press Ltd. Ontario Canada, pp 614–615 Johkura K, Usuda N, Tanaka Y, Fukasawa M, Murata K, Noda T, Ohno N (2022) Whole-cell observation of ZIO-stained Golgi apparatus in rat hepatocytes with serial block-face scanning electron microscope, SBF-SEM. Microscopy 71:262–270. Doi.org/10.1093/jmicro/dfac024/80/129 Benchimol M, De Souza W (1985) Tritrichomonas foetus : Cytochemical visualization of the endoplasmic reticulum-Golgi complex and lipids. Exp Parasitol 59:51–58. https://doi.org/10.1016/0014-4894(85)90056-6 Slomianny C, Prensier G (1990) A cytochemical ultrastructural study of the lysosomal system of different species of malarial parasites. J Protozool 37:465–470 Zuma AA, Barrias E, de Souza W (2021) Basic Biology of Trypanosoma cruzi. Curr Pharm Des 27:1671–1732 Urensen G, de Groot D (1974) Osmium-zinc iodide staining and the quantitative study of central synapses. Brain Res 74:131–142. 10.1016/0006-8993(74)90116-4 Morgado-Diaz JA, Agrellos OA, Dias WB, Previato JO, Mendonça-Previato L, De Souza W (2001) Isolation and characterization of the Golgi complex of the protozoan Trypanosoma cruzi . Parasitology 123:33–43. 10.1017/s0031182001007946 Docampo R (2024) Advances in the cellular biology, biochemistry, and molecular biology of acidocalcisomes. Microbiol Mol Biol Rev 88:e0004223. 10.1128/mmbr.00042-23 Souza-Melo N, de Souza GH, de Souza W (2025) Effects of TcFLA-1BP and TcGP72 Deletion on the Infectivity and Survival of Trypanosoma cruzi in Cell Culture. Int J Cell Biol. 10.1002/cbin.70076 Estevam H, Carvalho RT, Salgado LT, Alcantara CL, Aguar-Seabra J, de Souza W, Cunha e Silva NL, Pereira MG (2025) Cholesterol crystals in reservosomes of Trypanosoma cruzi . J Struct Biol 217(4):108259. 10.1016/j.jsb.2025.108259 Tachibana Y, Yamamoto M (2025) Recent advances in identifying and characterizing secretory proteins of Toxoplasma gondii by CRISPR-based screening. Parasitol Int 105. https://doi.org/10.1016/j.parint.2024.102997 Additional Declarations No competing interests reported. Supplementary Files answerMSZIOstainingT.cruzi.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 12 May, 2026 Reviews received at journal 11 May, 2026 Reviews received at journal 07 May, 2026 Reviews received at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers agreed at journal 22 Apr, 2026 Reviewers invited by journal 20 Apr, 2026 Editor assigned by journal 16 Apr, 2026 Submission checks completed at journal 16 Apr, 2026 First submitted to journal 15 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9430581","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":627969155,"identity":"bcd69c0f-0a52-4a61-bc9c-8873f4fd4b2b","order_by":0,"name":"Wanderley Souza","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA0UlEQVRIiWNgGAWjYPACmwQ2BsYHQJqBmUGCOC1pQC3MBkCaeC2HExigWhgIajE4f/iY5I+a83l87M0MzDwJ99gZpHsf4NdyIy1NQuLY7WI2nsMgLcXMDDLHDfBqMbvBYyZhwHY7sU0i/wAz748EoF/S8DvM7PwZM4mEf+eAWpJBthCj5UCOmcTBtgMkaLG/kZZs2diXDPbLwTlALWwyx/Brkew/fPDmj292efLtzYwP3iQkJPNLt+HXggIOAHEyGwkaIMCOZB2jYBSMglEw7AEApLA8esiYqawAAAAASUVORK5CYII=","orcid":"","institution":"Universidade Federal do Rio de Janeiro","correspondingAuthor":true,"prefix":"","firstName":"Wanderley","middleName":"","lastName":"Souza","suffix":""},{"id":627969156,"identity":"b22e389f-1859-448d-b5a2-56bf4ab6cab0","order_by":1,"name":"Noemia Rodrigues Alves","email":"","orcid":"","institution":"Universidade Federal do Rio de Janeiro","correspondingAuthor":false,"prefix":"","firstName":"Noemia","middleName":"Rodrigues","lastName":"Alves","suffix":""}],"badges":[],"createdAt":"2026-04-15 19:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9430581/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9430581/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108003814,"identity":"48983c9e-2353-4d5e-8eac-afd9d1860dbd","added_by":"auto","created_at":"2026-04-28 12:25:53","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":82527,"visible":true,"origin":"","legend":"\u003cp\u003e(A) control image of cells that were not incubated in the presence of the Zio-OsO4 solution. No labeling is seen in the various structures of the epimastigote form of \u003cem\u003eT. \u003c/em\u003ecruzi. N, nucleus; K, kinetoplast. G, Golgi complex; (B_C) low magnification of epimastigotes incubated in the presence of Zio-OsO4. The following structures are heavily labeled: the Golgi complex region (G) and profiles of the endoplasmic reticulum (ER). The nucleus and kinetoplast are not labeled.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9430581/v1/6ae4a100113ec5b8a2ac8d40.jpeg"},{"id":108003772,"identity":"a223c2de-2a63-4de8-a1c4-3a6292bbf650","added_by":"auto","created_at":"2026-04-28 12:25:48","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":81320,"visible":true,"origin":"","legend":"\u003cp\u003eDifferent views of labeled Golgi complex (G) and cisternae of the endoplasmic reticulum (ER) in different cells. Some Golgi complex cisternae show a variable labeling intensity. Fig. C shows intensely stained vesicles aligned to the trans region of the Golgi (arrows).\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9430581/v1/b211f18357fc5326d760e111.jpeg"},{"id":108003769,"identity":"da1a719d-fd7e-45fd-8614-6fb5a29c236e","added_by":"auto","created_at":"2026-04-28 12:25:48","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":90347,"visible":true,"origin":"","legend":"\u003cp\u003eCells are incubated in ZIO-OsO4 solution. (A) a reticular system, part of the ER, is labeled, leaving in between unlabeled vacuoles; (B) Tangential section of the region of emergence of the flagellum. Only some cisternae, localized around the flagellar pocket (FP), are labeled (arrows). They may correspond to part of the contractile vacuole. (C) In a few cells, the nuclear membrane was intensely labeled. (D) Labeling in a cisterna lining the region of adhesion between the cell body (CB) and the flagellum (F). (E), labeling of several vacuoles (V) and the reservosome R localized at the posterior region of the cell.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-9430581/v1/02b2e09f65a7762dd4095ccc.jpeg"},{"id":108007379,"identity":"e21564d8-07e0-4691-acb5-a33c13cb8026","added_by":"auto","created_at":"2026-04-28 12:59:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":397194,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9430581/v1/db612599-e6c4-4f09-a189-3082aa53e5a7.pdf"},{"id":108003793,"identity":"d7d8656a-e1f5-4838-939f-0b53b0f79279","added_by":"auto","created_at":"2026-04-28 12:25:50","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":17053,"visible":true,"origin":"","legend":"","description":"","filename":"answerMSZIOstainingT.cruzi.docx","url":"https://assets-eu.researchsquare.com/files/rs-9430581/v1/83929380d18c10294d1b42f1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Cytochemical localization of ZIO-stained compartments in Trypanosoma cruzi","fulltext":[{"header":"Introduction","content":"\u003cp\u003eClassical cytochemical methods adapted for transmission electron microscopy have made important contributions to the identification of structures and organelles in pathogenic protozoa, allowing the localization of carbohydrates on the cell surface, the characterization of organelles such as mitochondria, peroxisomes, endocytic pathways, and the lysosomal system, and the identification of lipid inclusions, among others. In some cases, these methods allow identification of the nature of the biochemical components analyzed, as in the localization of carbohydrates or enzymes. In other cases, the methods are more general in nature, only indicating whether the structures are made of lipids (as in the case of the use of osmium tetroxide/imidazole buffered solution [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] or basic proteins (as in the case of alcoholic phosphotungstic acid (PTA) [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Another interesting method is the use of impregnation by the Zinc-Osmium Tetroxide (ZIO) complex, initially introduced by Champy [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e] and Maillet [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] in light microscopy and adapted for transmission electron microscopy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] initially for visualization of synaptic vesicles of cholinergic junctions, without labeling other organelles located in the same place, such as mitochondria. Labeling was also found in part of the Golgi complex, especially in the GERL and lysosomes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Later, Pellegrino de Iraldi and co-workers, and other authors found labeling in the endoplasmic reticulum, part of the Golgi complex, and some mitochondria [\u003cspan additionalcitationids=\"CR10 CR11 CR12 CR13 CR14\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Although the biochemical nature of the molecules that interact with ZIO remains unclear, there are indications of interactions with lipoprotein complexes, calcium-binding sites, and even proteins containing sulfhydryl groups [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. More recently, Jokhura et al. [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] used ZIO impregnation for three-dimensional reconstruction of the Golgi complex in rat hepatocytes.\u003c/p\u003e \u003cp\u003eTo date, only two articles have described results obtained with ZIO in pathogenic protozoa. Benchimol and De Souza [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] showed labeling in the endoplasmic reticulum-Golgi complex, nuclear pore, and reticulum profiles located near the region of adhesion of the recurrent flagellum to the body of the protozoan \u003cem\u003eTritrichomonas foetus\u003c/em\u003e. Then Slomianny and Prensier [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] demonstrated labeling of the endoplasmic reticulum, Golgi complex, and lysosomal systems in \u003cem\u003ePlasmodium falciparum.\u003c/em\u003e\u003c/p\u003e \u003cp\u003eAlthough the structural organization of \u003cem\u003eT. cruzi\u003c/em\u003e has been well established by conventional transmission electron microscopy of thin sections [Review in 19], there is little information on differential labeling of cell structures using a cytochemical approach. In this article, we describe the results obtained with ZIO for \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e, a pathogenic protozoan that causes Chagas disease, a relevant human disease, and that presents a distinct cellular organization. The results show variation in labeling of the various structures and organelles, possibly indicating heterogeneity in their composition throughout the cell cycle.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eObtaining the parasites\u003c/h2\u003e \u003cp\u003e \u003cem\u003eT. cruzi\u003c/em\u003e epimastigotes were maintained in LIT culture medium supplemented with 10% of fetal bovine serum. After 4 days of inoculation, when parasite density reached 10\u003csup\u003e6\u003c/sup\u003e cells/ml, the parasites were collected by centrifugation.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eZinc iodide-osmium technique\u003c/h3\u003e\n\u003cp\u003eThe same protocol was applied to both cell types. Fixation was carried out in 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 2 hours. Cells were rinsed in a solution of 8,5% sucrose in 0.1 M phosphate buffer, pH 7,2, 2 times for 10 minutes each. After that, the cells were rinsed with Tris aminomethane buffer (1,13M NaCl; 0,011M CaCl2; 0,03M MgCl2; 0,01M Tris aminomethane [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. The final pH of the incubation medium was adjusted to 3.8\u0026ndash;4.5 with 1N HCl. The cells were then incubated in ZIO solution for 18 hours, at 4\u0026deg;C in the dark. Shortly before the incubation, the ZIO solution was prepared as follows: 3 g Zn powder and 1 g iodine resublimed was suspended in 10 ml distilled water, separately. These solutions were mixed in a ratio of 1:1. After filtering (in a 0.45 \u0026micro;m Millipore filter placed in a syringe), 4 ml of this solution was mixed with 4 ml of the Tris aminomethane buffer. Immediately before using 4 parts of this solution were mixed with 1 part of a 2% OsO4 aqueous solution. After dehydration in a graded series of acetone, cells were embedded in an epoxy resin, sectioned, and observed by transmission electron microscopy.\u003c/p\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eIn general, procedures for preparing biological specimens using metal impregnations require long incubation times. Therefore, the quality of fixation is slightly lower than that obtained by conventional methods. However, after several attempts, we established a fixation and staining procedure that proved reproducible across several experiments. Figure\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eA shows an image of the epimastigote form of \u003cem\u003eT. cruzi\u003c/em\u003e processed in a similar way to the cells impregnated with ZIO, but without passing through this reagent. The image clearly shows the nucleus, the kinetoplast, and the Golgi complex. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-E show equivalent areas of epimastigotes impregnated with ZIO, where a clearly marked region of the Golgi complex can be seen, as well as a set of vacuoles located in the posterior region and corresponding to the endolysosomal system, consisting of the reservosomes, as well as other structures described below. Figure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003e. (A) control image of cells that were not incubated in the presence of the Zio-OsO4 solution. No labeling is seen in the various structures of the epimastigote form of \u003cem\u003eT.\u003c/em\u003e cruzi. N, nucleus; K, kinetoplast. G, Golgi complex; (B_C) low magnification of epimastigotes incubated in the presence of Zio-OsO4. The following structures are heavily labeled: the Golgi complex region (G) and profiles of the endoplasmic reticulum (ER). The nucleus and kinetoplast are not labeled.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eCertainly, one of the most marked structures is the Golgi complex. However, not all the cisternae and vesicles that form this organelle were marked in the same way. In some cells, all cisternae on the cis and trans faces were marked, with the marks more pronounced in more expanded lateral regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, E). In some cells, the label was more evident in the first cisternae (Cis face), although not in their entire extent, whereas in other cells it was evident in the last cisternae of the trans face. Images such as Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e2\u003c/span\u003eC clearly show a non-uniform Golgi mark, with densely marked cisternae associated with dense vesicles that detach from the central cisterna. Some cells showed the electron-dense product throughout the Golgi as well as in the cisternae of the endoplasmic reticulum distributed throughout the anterior region, close to the Golgi, and in the posterior region, surrounding the reservosomes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and E). These data clearly show that the system of cisternae and vacuoles associated with the Golgi complex of \u003cem\u003eT. cruzi\u003c/em\u003e is highly heterogeneous, indicating a dynamic activity of the glycoprotein synthesis and glycosylation system. This information is relevant because there are no detailed studies of the Golgi complex in this protozoan, although the organelle has been isolated by cell fractionation [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Further three-dimensional reconstruction of stained cells may add more information on this topic.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe labeling on other sites deserves to be highlighted. First, a reticular system located in the lateral region of the flagellar pocket, possibly formed by cisternae and vesicles that form the contractile vacuole of \u003cem\u003eT. cruzi\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This compartment concentrates several ions and may also act as a transport route for components of the Golgi complex that are secreted into the interior of the flagellar pocket [Review in 22). Second, a reticular structure located just below the portion of the plasma membrane of the protozoan in the area close to the flagellum (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). It is possible that this component is associated with the FAZ (Flagellar Attachment Zone) complex, which contains a set of important proteins such as GP 72 [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Third, structures are located within the endocytic system, especially in the reservosome. Based on what we know about the reservosome, it is possible that these structures correspond to the lipid component, which tends to form crystalline lipid structures [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Finally, we note that some epimastigotes exhibit strong nuclear membrane staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBoth authros were involved in the experiments, microscopy observation and writting the article.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe authors thank all members of the laboratory for technical help and discussion. This work has been supported by Conselho Nacional de Desenvolvimento Cient\u0026iacute;fico e Tecnol\u0026oacute;gico-CNPq, Funda\u0026ccedil;\u0026atilde;o de Apoio \u0026agrave; Forma\u0026ccedil;\u0026atilde;o de Pessoal de N\u0026iacute;vel Superior-CAPES, Financiadora de Estudos e Projetos-FINEP, and Funda\u0026ccedil;\u0026atilde;o Carlos Chagas Filho de Apoio \u0026agrave; Pesquisa do Estado do Rio de Janeiro-FAPERJ\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSoares MJ, de Souza MF, de Souza W (1987) Ultrastructural visualization of lipids in trypanosomatids. 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Parasitol Int 105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.parint.2024.102997\u003c/span\u003e\u003cspan address=\"10.1016/j.parint.2024.102997\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"ZIO-OsO4 staining, Electron microscopy, Trypanosoma cruzi","lastPublishedDoi":"10.21203/rs.3.rs-9430581/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9430581/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElectron microscopy cytochemistry has contributed to a better understanding of the identification of some cytoplasmic components of eukaryotic cells, even when they are not highly specific, yet they still allow identification of cell components. Here, we describe results obtained by impregnating cells with a Zinc-Osmium Tetroxide complex to label certain organelles of \u003cem\u003eTrypanosoma cruzi\u003c/em\u003e. Labeling of some cisternae of the Golgi complex and components of the endocytic pathway, especially the reservosomes, was observed. Labeling of ER cisternae, a reticular structure located just below the portion of the plasma membrane associated with the flagellar attachment zone (FAZ), and the contractile vacuole, was observed. Variation in the intensity of the labeling pattern is probably related to the dynamics of the cell cycle.\u003c/p\u003e","manuscriptTitle":"Cytochemical localization of ZIO-stained compartments in Trypanosoma cruzi","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-28 12:25:22","doi":"10.21203/rs.3.rs-9430581/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-12T09:45:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T21:04:10+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T04:41:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-22T19:28:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"41520746506223707582995617378557363054","date":"2026-04-22T13:31:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268913270734868120545446920250802289143","date":"2026-04-22T11:36:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"74576444629159516936693364944487487191","date":"2026-04-22T10:57:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-20T10:06:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-16T14:08:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-16T14:08:13+00:00","index":"","fulltext":""},{"type":"submitted","content":"Acta Parasitologica","date":"2026-04-15T19:14:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"acta-parasitologica","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"actp","sideBox":"Learn more about [Acta Parasitologica](http://link.springer.com/journal/11686)","snPcode":"11686","submissionUrl":"https://submission.springernature.com/new-submission/11686/3","title":"Acta Parasitologica","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f9800d4f-b537-4ca7-a20c-06e11754fde1","owner":[],"postedDate":"April 28th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-12T09:45:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-11T21:04:10+00:00","index":12,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T04:41:03+00:00","index":11,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-12T10:43:58+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-28 12:25:22","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9430581","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9430581","identity":"rs-9430581","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}