Mouse anti flag 1e6

Third instar larval fillet was fixed with 3.7% formaldehyde for 20 min at room temperature. After brief washing with 0.3% PBT (PBS containing 0.3% Triton X-100), dissected tissues were incubated in blocking buffer (5% normal donkey serum in 0.3% PBT) for 50 min at room temperature. The tissues were incubated at 4°C with primary antibody. The following primary antibodies were used in this study: rat anti-HA (3F10, Roche; 1:200 dilution) for detecting the HA epitope; mouse anti-V5 (R960-25; Thermo Fisher Scientific; 1:200) for detecting the V5 epitope; and mouse anti-Flag (1E6, Wako; 1:200) for detecting the Flag epitope. To detect primary antibodies, the following secondary antibodies were used: donkey anti-mouse cy3 (Jackson Immunoresearch Laboratories; 1:300), goat anti-mouse Alexa Fluor 647 (Invitrogen; 1:1,000), goat anti-HRP Alexa Fluor 488 (Jackson Immunoresearch Laboratories; 1:1,000), and goat anti-rat Alexa Fluor 647 (Jackson Immunoresearch Laboratories; 1:1,000).