Virginie Orgogozo
Antibody staining of larval gonad and other tissues
Staging larvae - Dissection
Let flies lay eggs for 4h/ON.
About 64h / three days later, select L2 larvae and put them in a new agar plate with yeast paste.
Every 2h, select the L3 larvae from this agar plate. The L2->L3 molt
should be around 72h. Select only female larvae (much smaller gonad).
Dissection
Pin down the larvae tail in PBT (PBS+0.1% Tween-20) on a Sylgard
dissection plate by inserting pins on the head and then the tail
Cut in the middle of the larvae anterior to the gonad and then along the midline in the posterior part of the body.
Select a piece of the lateral fat body associated with the gonad. Put it in the little basket in PBT on ice
Fixation, Coloration
Fix 20min in 5% FA+ EDTA (900uL 5% FA + 100uL EDTA 0.5M)
Incubate in methanol for 3-5min
Quick wash in PBT
PBT 30min
PBT, 0.1% BSA, 2% GS (20uLGS, 5uL BSA 20% in 1mL PBT) 30min
Primary antibodies ON at 4°C or 2h at RT (diluted in PBT+BSA+GS)
3 washes 15min each in PBT
PBT, 0.1% BSA, 2% GS 1h
Secondary antibodies 3h at RT (diluted in PBT)
3 washes 15min each in PBT
Hoechst (Ryan’s tube) dilution 1/1000 in PBT 45min
3 washes 15min each in PBT
Mounting
Put 55uL of Vectashield mounting medium on a slide
Take the fat body+ovaries with forceps and put them in the Vectashield medium, remove the fat body
cover with coverslip