Immunohistochemistry

We dissected legs from the fly using forceps.  We transferred the legs to a 0.6mL microcentrifuge tube containing fixative: 4% paraformaldehyde in phosphate-buffered saline (PBS). We fixed the legs for 20 minutes. 

After fixation, we washed the legs three times with PBST (PBS + Triton-X, 0.2% w/w), then blocked them for 20 minutes in PBST + 5% normal goat serum (Sigma Aldrich G9023). 

To stain the biocytin-filled cells with a phalloidin co-label, we incubated the legs in 500µL PBT with sodium azide (0.01%, Thermo Fisher 190380050), Phalloidin Alexa Fluor 647 (1 unit, Thermo Fisher A22287) and Streptavidin Alexa Fluor 568 conjugate (1:250, Thermo Fisher S11226). We incubated the legs in stain for two weeks at 4°C, with occasional nutation.

Following staining, the leg was mounted in Vectashield (Vector Labs H-1900), using double-stick tape as spacers.