Materials and Reagents

Equipment

Procedure

Dissect larvae in 1:10 solution of PBS. Larval dissection can be accomplished by pulling the larvae apart at approximately the A4 segment and inverting the body cavity such that the wing disc is visible. Note, wing disc must be exposed to medium in order to ensure consistent staining.
Fix dissected larvae for 30min in 1.5mL Eppendorf tube with 4% paraformaldehyde. Place tube on nutator.
Remove 4% paraformaldehyde from tube and replace with a 1:10 solution of PBS. Place tube on nutator for 10min.
Remove 1:10 solution of PBS from tube and replace with 0.5% PBTX. Place tube on nutator for 5min.
Remove 0.5% PBTX and add 1:1000 solution of Hoechst DNA stain (dilution created with Hoechst and 0.1% PBTX). Place tube on nutator 3-4min.
Remove Hoechst and wash sample with 0.1% PBTX for 10min. Repeat wash twice more (total of 3 washes).
Add primary antibody solution to tube and move flies to a nutator at ~4 degrees celsius. Larvae should be left on nutator overnight (~11h).
Remove primary antibody solution. Add 0.1% PBTX to tube for three 10min washes on nutator.
Add secondary antibody to tube and wrap the tube in aluminum foil to mitigate light exposure. Place tube on nutator for 2h.
Remove secondary antibody and add 0.1% PBTX for three 10min washes on nutator.
Mount wing disc on a microscope slide. Add a drop of Fluoromount-G on wing discs before gently placing a coverslip on the slide. Note, larvae can be easily removed from Eppendorf by cutting the tip off of a p200 micropipette tip.

Recipes