All used transgenic constructs were crossed into a cn bw background [38] (link) to eliminate eye pigmentation. Frozen sections of adult heads were performed using a cryostat microtome (Zeiss) and deposited on superfrost Plus slides (Fisher), as previously described [43] (link). For immuno Histochemistry conditions were the same as above. For X-Gal reactions, Horizontal eye sections were fixed 5±10 min in PBS 0.25% gluteraldehyde. They were stained in a solution of 7.2 mM Na2HPO4, 2.8 mM NaH2PO4, 150 mM NaCl, 1 mM MgCl2, 3 mM K3[Fe(CN)6], 3 mM K4[Fe(CN)6], containing a 1/30 dilution of X-Gal (30 mg/ml in dimethyl formamide). After washing in PBS, slides were mounted in aquamount (Lerner Laboratories, Fisher).
Cryostat microtome
Manufactured by Zeiss
The Cryostat Microtome is a laboratory instrument used for cutting thin, frozen tissue sections. It maintains the sample at a constant, low temperature to enable the preparation of high-quality samples for microscopic analysis.
Automatically generated - may contain errors
2 protocols using cryostat microtome
1
Cryosectioning and X-Gal Staining of Fly Eyes
2
BrdU Labeling and Immunohistochemistry
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Manually-dechorionated embryos were labeled with BrdU (Roche) by incubating them for 1 h 30min on ice in a solution of 10 mM BrdU and 15% DMSO in EM at 8 dpf. The embryos were then placed in EM, incubated for 1 h at 28.5°C, and fixed using 4% paraformaldehyde in PBS. Embryos were processed for immunohistochemistry, treated for 20 min with 2 M HCl, and were then processed for anti-BrdU immunohistochemistry. For immunohistochemistry, we used the following primary antibodies: mouse anti-BrdU (G3G4, 1:250, Developmental Studies Hybridoma Bank), rabbit anti-Sox10 (1:800, Lifespan bioscience), rat cd11b (1:100, BD bioscience), mouse anti-sodium channel (1:500, Sigma), and mouse anti-acetylated tubulin (1:1000, Sigma). Alexa 488-, 568-, 647-conjugated secondary antibodies were used for fluorescence detection (1:500, Molecular Probe). For histology section, embryos were embedded in 1.5% agar/5% sucrose. Sections (10 μm) were obtained by using a cryostat microtome (Zeiss).
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