Immunohistochemical Analysis of Brain Samples

Immunohistochemistry follows protocols previously reported^{15 (link),16 ,18 (link)}. Mice were overdosed with 3% isoflurane and perfused transcardially with cold (4 °C) phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA) in PBS. Brains were extracted and stored overnight in PFA at 4 °C. Fifty μm coronal sections were collected in serial order using a vibratome and collected in cold PBS. Sections were blocked for 1 hour at room temperature in PBST and 5% normal goat serum (NGS) or Bovine albumin serum (BSA) on a shaker. Sections were transferred to wells containing primary antibodies (1:1000 guinea anti-c-Fos [SySy]; 1:1000 rabbit anti-RFP [Rockland]; 1:5000 chicken anti-GFP [Invitrogen]) and allowed to incubate on a shaker overnight at room temperature or 3 days at 4 degrees C. Sections were then washed in PBST for 10-min (x3), followed by 2-hour incubation with secondary antibody (1:200 Alexa 555 anti-rabbit [Invitrogen]; 1:200 Alexa anti-guinea 647 [Invitrogen]; 1:200 Alexa 488 anti-chicken [Invitrogen]). Following three additional 10-min washes in PBST, sections were mounted onto micro slides (VWR International, LLC). Vectashield Hart Set Mounting Medium with DAPI (Vector Laboratories, Inc) was applied, slides were coverslipped, and allowed to dry overnight.