Mouse anti dlg 4f3

Third instar larvae were dissected in ice-cold 1xPBS and fillets were fixed in 4%PFA in PBS at room temperature for 30min. For Futsch staining, fillets were fixed in ice-cold methanol for 10min at −20°C. Samples were washed in 1xPBS for 10 min followed by two washes in 1xPBST (PBS + 0.1% Triton) for 10min each. Fillets were transferred to 0.5μl tubes for primary antibody incubation overnight at 4°C with agitation. After three 15 min washes in 1xPBST, samples were incubated in secondary antibodies for 1 h at room temperature. Samples were then washed three times for 10 min each and mounted with Fluoromount-G DAPI (SouthernBiotech) or SlowFade Glass Antifade Mountant (Invitrogen). Mouse anti-Dlg (4F3) 1:1000, anti-Futsch (22C10) 1:100, and anti Brp (nc82) 1:100 were obtained from Developmental Studies Hybridoma Bank (DSHB, University of Iowa). Rabbit anti- GluRIIC (1:5000) was a kind gift from Aaron DiAntonio. Alexa 647-conjugated goat anti-HRP was used at 1:200 (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA). Goat anti-mouse Alexa 488 (Invitrogen) was used at 1:400 while goat anti-mouse TRITC and anti-rabbit FITC (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) were used at 1:400.