Coronal sections were prepared on a cryostat at 20 μm and immediately mounted on slides. Slides were maintained at −80°C until ready for staining. Every 10th section was Nissl stained with Cresyl Violet to identify sections corresponding to areas of interest (Franklin and Paxinos, 2013 ).
Immunostaining was done as described previously (Beaudoin et al., 2012 (link)). The following primary antibodies were used mouse anti-bassoon (1:400, ABCAM ab82958), rabbit anti-gephyrin (1:1000, ABCAM ab32206), rabbit anti-N-methyl-d-aspartate (NMDA) receptor subunit 1 (1:1000, Thermo Fisher Scientific PA3-102), and chicken anti-tyrosine hydroxylase (TH) (1:500, ABCAM ab76442). Secondary antibodies used were goat anti-mouse (1:1000 for all; Alexa568, ABCAM ab175473; Alexa488, Fisher A11029; Alexa647, Fisher A21241), goat anti-rabbit (1:1000 for all; Alexa488 ABCAM ab150077; Alexa568, Fisher A11036), and/or goat anti-chicken (1:1000, Alexa405, ABCAM ab175675). Coverslips were mounted with ProLong Gold antifade (Invitrogen).
Immunostaining was done as described previously (Beaudoin et al., 2012 (link)). The following primary antibodies were used mouse anti-bassoon (1:400, ABCAM ab82958), rabbit anti-gephyrin (1:1000, ABCAM ab32206), rabbit anti-N-methyl-d-aspartate (NMDA) receptor subunit 1 (1:1000, Thermo Fisher Scientific PA3-102), and chicken anti-tyrosine hydroxylase (TH) (1:500, ABCAM ab76442). Secondary antibodies used were goat anti-mouse (1:1000 for all; Alexa568, ABCAM ab175473; Alexa488, Fisher A11029; Alexa647, Fisher A21241), goat anti-rabbit (1:1000 for all; Alexa488 ABCAM ab150077; Alexa568, Fisher A11036), and/or goat anti-chicken (1:1000, Alexa405, ABCAM ab175675). Coverslips were mounted with ProLong Gold antifade (Invitrogen).