Perfusion, Fixation, and Sectioning of Rat Brains

Rats were deeply anesthetized with isoflurane and transcardially perfused with ice-cold PBS, and followed by ice-cold 4% PFA (paraformaldehyde) in PBS. Brains were fixated in PFA overnight and transferred to 30% sucrose in PBS to equilibrate for three days as previously described [6 (link), 7 (link), 9 (link)]. Following this, 20 µm coronal sections were collected by using Leica CM3050s cryostat (Leica Biosystems, Germany) and washed with PBS for 10 min. Sections were washed in PBS and coverslipped with Vectashield mounting medium. Sections were made after viral transfer for opsin verification, and these were stained with anti-rabbit GFP (1:500, #AB290, Abcam, USA). Secondary antibody was anti-rabbit immunoglobulin G (Ig G) conjugated to Alexa Fluor 488 (1:500, Life Technologies, USA). Images were acquired with a Zeiss LSM 700 Confocal Microscope (Carl Zeiss, Thornwood, NY, USA). Images containing cannulas were stained with cresyl violet and examined at 10 × magnification with an Axio Zoom widefield microscope (Carl Zeiss, Thornwood, NY, USA).