Immunofluorescent Labeling of Drosophila Pupa Abdomen

Dorsal abdomen specimens were dissected from pupae between the P14 and P15(i) developmental stages [12 (link),16 (link),50 ]. Male and female specimens were combined, and females were distinguished by the removal of their wings. The ensuing steps were done with male and female specimens together in the same tubes or plate in order to expose to identical conditions. Specimens were fixed for 35 min in PBST solution (phosphate buffered saline with 0.3% Triton X-100) with 4% paraformaldehyde (Electron Microscopy Services). After fixation, specimens were washed twice with PBST and then blocked for 1 hour at room temperature in a blocking solution (PBST with 1% Bovine Serum Albumin). Specimens were then incubated overnight at 4°C with a primary antibody in PBST. These were either mouse monoclonal anti-Abd-B (Developmental Studies Hybridoma Bank, 1A2E9) at a dilution of 1:200 of a concentrated stock, rabbit anti-Bab1 primary antibody [11 (link)] at a 1:200 dilution, and rabbit anti-Trx [39 (link)] at a 1:200 dilution.
After four washes with PBST and then 1 hour in blocking solution, specimens were incubated with either goat anti-mouse Alexa Fluor 647 (Invitrogen, #A21236) secondary antibody, or goat anti-rabbit Alexa Fluor 646 (Invitrogen, #A21244) secondary antibody. These secondary antibodies were used at a 1:500 dilution in PBST, and incubated for 2 hours at room temperature. Following four washes with PBST, the specimens were equilibrated for ten minutes at room temperature in Glycerol Mount:PBST (50% glycerol, 50% PBST) solution. Specimens were then transferred to the glycerol mount (80% glycerol) before being situated between a glass cover slip and slide for imaging with a confocal microscope. Cover slip and slides were separated by one piece of double-sided sticky tape, for which a hole was cut out in the center by a razor blade. Specimens are situated in the hole with their cuticle towards the cover slip side.