Alexa fluor 633 goat anti mouse lgg

Dissected brains were fixed with 4% paraformaldehyde for 15 min at room temperature. Fixed tissue was blocked with PBS containing 2% Triton X-100 and 10% normal goat serum (NGS) for 30 min and then incubated for 1 day each (with washing in between) at room temperature in PBS containing 1% Triton X-100, 0.25% NGS, and a primary antibody or secondary antibody solution. The brain was mounted in 20 μl Vectashield medium. We used the following primary antibodies at the indicated dilutions: 1:50 mouse anti-bruchpilot (nc82) (Developmental Studies Hybridoma Bank, Iowa), 1:1000 rabbit anti-5HT (Sigma, S5545). Secondary antibodies from Invitrogen were used at dilutions of 1:250, which were Alexa Fluor 633 goat anti-mouse lgG (Life Technologies, A21050), Alexa Fluor 568 goat anti-mouse lgG (Life Technologies, A11004), Alexa Fluor 633 goat anti-rabbit (Invitrogen, A21071). Confocal images were acquired with Zeiss LMS710 confocal laser scanning microscope under 40x or 63x magnification.