Goat anti rat alexa fluor 647

Larvae (120 hr AEL), pupae (18 hr APF), and adult flies (10d adult and 40d adult) were dissected in 1x Phosphate Buffered Saline (PBS) to obtain fillet or brain samples for immunohistochemical analyses. Obtained samples were fixed in 4% Paraformaldehyde for 20 min, washed in 0.3% PBST (Triton-X100 0.3% in PBS), and blocked in blocking buffer (5% Normal donkey serum or normal goat serum in 0.3% PBST) for 45 min at room temperature. Samples were then incubated with the following primary antibodies for overnight at 4°C: mouse anti-Flag (1F6, Wako; 1:400 dilution), rat anti-HA (3F10, Roche; 1:200 dilution), rabbit anti-TBPH (1:100 dilution) (Lin et al., 2011 (link)), and goat anti-HRP Alexa Fluor 488 (Jackson Immunoresearch Laboratories; 1:400 dilution) antibodies. The next day, samples were washed for 10 min (repeated three times) in 0.3% PBST and incubated with the following secondary antibodies for 4 hr: goat anti-mouse Alexa Fluor 647 (Invitrogen; 1:400 dilution), goat anti-rat Alexa Fluor 647 (Jackson Immunoresearch Laboratories; 1:200 dilution), and goat anti-rabbit Alexa Fluor 647 (Invitrogen; 1:400 dilution) antibodies. Samples were then rinsed with 0.3% PBST for 10 min (repeated three times) and mounted with 70% glycerol in phosphate buffered saline (PBG) for imaging.