Fixation and staining of Drosophila L1 larval brains for immunofluorescence microscopy and preparation for live imaging

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This paper details a protocol for preparing <italic>Drosophila</italic> L1 larval brains for immunofluorescence microscopy and live imaging, including methods for embryo collection, fixation, staining, and 3D printed slides.

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AI-generated deep summary by claude@2026-07, 2026-07-16 · read from full text

This protocols.io paper describes a step-by-step method for fixing, staining, and mounting Drosophila L1 larval brains for immunofluorescence microscopy, as well as preparing brains for live imaging. The authors outline high-level workflow components such as sample handling, fixation and staining procedures, and preparation steps aimed at preserving tissue integrity for imaging. A major caveat is that the protocol is specific to Drosophila L1 larval brains and depends on careful adherence to the stated reagents and imaging requirements to achieve usable signal. The paper does not explicitly discuss endometriosis or adenomyosis; it was included in the corpus via a keyword match in the upstream search index.

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Abstract

Abstract Drosophila first instar (L1) larval brains (LBs) contain frequent quiescent neural stem cells (qNSCs) as well as activated neuroblasts, making them favorable for studying stem cell quiescence and activation. However, the small size of LBs at the L1 stage necessitates the use of modified methods to prepare the LBs for immunofluorescence microscopy (IFM). The protocol described here allows efficient collection of embryos and maturation of larvae to the mid-L1 stage, followed by dissection, fixation and processing of LBs through the antibody staining steps for IFM. The entire procedure can be completed in ~3-5 days. Methods are also described for use in preparing L1 brains for live imaging experiments, including a file to create accessible and cost-effective 3D printed slides that can be fit with an O2-permeable membrane for live imaging.
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