Alexa 647 conjugated donkey anti goat secondary antibody

Mice were transcardially perfused with 4% paraformaldehyde and then postfixed overnight. Coronal brain slices containing the LC or dorsal raphe were cut at a thickness of 50 μm, using a vibratome (Leica VT1000S). Free-floating slices were permeabilized with 0.5% Triton-X 100 and blocked with 1% fish gelatin in 0.1 M phosphate-buffered saline (pH 7.4). NE-positive neurons were immunostained by incubating overnight with rabbit anti-dopamine beta-hydroxylase (1:500, Abcam ab209487) and labeled with Alexa 546-conjugated donkey anti-rabbit secondary antibody (1:1000, Invitrogen). 5-HT neurons were immunostained by incubating with goat anti-5-HT antibody (1:500, Abcam ab66047) and similarly labeled with Alexa 647-conjugated donkey anti-goat secondary antibody (1:1000, Invitrogen).

Mice were transcardially perfused with 4% paraformaldehyde, then postfixed overnight. Coronal brain slices containing the LC or dorsal raphe were cut at a thickness of 50 μm, using a vibratome (Leica VT1000S). Free-floating slices were permeabilized with 0.5% Triton-X 100 and blocked with 1% fish gelatin in 0.1 M phosphate-buffered saline (pH 7.4). NE-positive neurons were immunostained by incubating overnight with rabbit anti-dopamine beta hydroxylase (1:500, Abcam ab209487) and labeled with Alexa 546-conjugated donkey anti-rabbit secondary antibody (1:1000, Invitrogen). 5-HT neurons were immunostained by incubating with goat anti-5-HT antibody (1:500, Abcam ab66047) and similarly labeled with Alexa 647-conjugated donkey anti-goat secondary antibody (1:1000, Invitrogen).