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- Dissect imaginal disc (late third instar) as normal. i.e. invert anterior half of larvae, remove fat body and salivary glands.
- Tissues are fixed in 4% paraformaldehyde for 5 min at RT. Any longer can denature too much of the GFP. I have seen some recommendations for 1% paraformaldehyde, but have not tried this yet. See note below
- Tissues rinsed 3 times in PBT and then washed in PBT for 10 min.
- Tissues placed in Anti-fade Mountant. If nuclear co-stain is needed, use with Hoechst (33342) added to a 1:15000 dilution (1:10000 and even 1:1000 works as well depending on application).
- Tissues vessels wrapped with foil. After 5 min incubation the imaginal disc are dissected on a slide.
- Do not seal with nail polish as this may quench the GFP. So slide must be treated carefully.
Remember that paraformaldehyde should be made fresh every 6 months (or else it produces chemical byproducts that quench GFP). Once a stock of 4% PF is made in a batch, freeze small aliquots. Whole batch should be replaced every 6 months. Check recipe in molecular lab!
Note: It may be the paraformaldehyde based changes to GFP are actually reversible. See this paper. i.e. treatment with alkaline buffer solutions (such as 0.1 M Na2CO3, pH=11.6).