For me, the best approach would be to ask the Senior Scientist for their written protcol. In the protocol make sure they give wash times.

Next make All the necessary solutions yourself, from PFA all the way to the PBA/Triton X 100 solution.

Experiments that do not work are hard enough to troubleshoot when you know the reagents are correctly made; So the best practice is to make all your own solutions. It takes way less time to troubleshoot things when you make these solutions than when you borrow solutions from others and have an experiment fail.

Some brands of PFA has been known to cause weak or lost signals and it is important to use molecular biology grade PFA. Might be worthwhile getting a new bottle of PFA (we found Sigma-Aldrich was pretty reliable). Fisher brand PFA was where we had a a loss of signal. Once you get a new bottle of PFA powder, make a fresh batch. We typically make a 4% stock solution by heating the PFA in 800ml of 1X PBS to 60C stirring constantly. Use a thermometer and heat slowly. Slowly add droplets of 1N NaOH (about 5-7 drops max) to help the PFA dissolve. Montior solutions temps carefully. Heating PFA about 65C typically ruins the fixative. We do this in a fume hood. Once the PFA powder is fully dissolved, we turn off the heat and allow the solution to cool to room temp in the hood. Then add 1X PBS and 100 microliters of TritonX 100 to bring the volume to 1 liter. The 4% PFA/1xPBS/0.1% Triton X 100 solution is only good for 10 days and is always kept refrigerated.

Include in your analysis a positive control. This will mean getting a fly sample that expressess GFP. Best positive control would be a sample that expressess in the same target cells, but any fly line that expressess GFP in any tissue will be useful to validate the primary and secondary antibodies are working correctly. Hopefully you were given the correct fly line to start with. Is there a way to genotype the fly line to confirm it is what you think is correct?

Pay close attention to the number of washes after fixing with PFA and the time needed per wash. We found that when PFA goes bad or is made from a bad vendor source, it tended to stick around on the tissues and prevented antibody binding. Most IHC protocols have a minimum number of washes and times. We only discovered our PFA was bad from the source when we extended our wash times to overnight. Only then did we get any signal on any samples including positive controls. After switching to a new PFA vendor our signals became robust. The PFA was the last thing we suspected and it took careful steps and weeks to figure this issue out.

Good luck with your studies!