Immunohistochemistry

Immunostaining of brain was performed as described before with some modifications.^49,62 Animals were anesthetized with sodium pentobarbital and transcardially perfused with 15 ml of phosphate buffered saline (PBS) (Sigma-Aldrich) followed by 30 ml of 4% w/v paraformaldehyde (PFA) (Fischer Scientific) in PBS. Brains were extracted, stored overnight in in 4% PFA in PBS at 4 °C, then in 15% w/v sucrose in PBS for 8 hr at 4 °C, followed by 24 hr in 30% sucrose, and then in 60% sucrose until sectioning. The tissue was then frozen and cut into 20-µm thick coronal sections on a cryostat, mounted onto microscope slides and kept at −80°C. Brain sections were permeabilized with 0.5% Triton X-100 (Sigma) in PBS (PBST) for 30 min, blocked with 10% normal donkey serum (NDS) in 0.1% PBST for 30 min and incubated in 5% NDS-0.1% PBST at 4 °C overnight with the following primary antibodies against NOS1, GFP, GAD67, or CaMKII. The next day, after three washes with PBS, specimens were incubated with anti-chicken Alexa flour 488-, anti-rabbit Cy3- or anti-mouse Alexa flour 647-conjugated donkey secondary antibodies (1:200; Jackson ImmunoResearch Laboratories). Then, sections were washed with PBS and mounted using an anti-fade mounting medium that contained DAPI (Vetashield, H-1200, Vector Laboratories). Fluorescent images were collected using a confocal microscope (Leica TCS SP8) with a 20×/0.75 NA objective (11506517, Leica). The following primary antibodies were used: rabbit anti-NOS1 polyclonal antibody (1:300, AB5380, Sigma-Aldrich), chicken anti-GFP polyclonal antibody (1:500, A10262, Invitrogen), mouse anti-GAD67 monoclonal antibody (1:300, MAB5406, Sigma-Aldrich), rabbit anti-CaMKII monoclonal antibody (1:300, ab52476, abcam).