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

<title>Abstract</title> <italic>Drosophila</italic> 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.