Anti rabbit immunoglobulin g igg conjugated to alexa fluor 488

Rats were anesthetized deeply with isoflurane and transcardially perfused with ice-cold PBS. Brains were fixed in paraformaldehyde (PFA) overnight and then transferred to 30% sucrose in PBS to equilibrate for 3 days as described (Lee et al., 2015 (link)). Coronal sections, 20 μm, were washed in PBS and coverslipped with Vectashield mounting medium. Images containing tetrodes were stained with cresyl violet and viewed and recorded under a Nikon Eclipse 80i microscope with a DS-U2 camera head. Sections also were made after viral transfer for opsin verification, and these sections were stained with anti-rabbit GFP (1:500, #AB290, Abcam), CaMKII-α (6G9) mouse monoclonal antibody (mAb) (1:100, #50049, Cell Signaling Technology), and DAPI (1:200, Vector Laboratories) antibodies. Secondary antibodies were anti-rabbit immunoglobulin G (IgG) conjugated to Alexa Fluor 488, and anti-mouse IgG conjugated to Alexa Fluor 647 (1:200, Life Technologies). Images were acquired with a Zeiss LSM 700 confocal microscope (Carl Zeiss).

Rats were deeply anesthetized with isoflurane and transcardially perfused with ice-cold PBS and paraformaldehyde (PFA). After extraction, brains were fixed in PFA overnight and then cryoprotected in 30% sucrose in PBS for 48 hours or until sinking [60 (link)]. 20 μm coronal sections were washed in PBS and coverslipped with Vectashield mounting medium. Sections from brains containing tetrodes were stained with cresyl violet and imaged at 10x magnification with an Axio Zoom widefield microscope (Carl Zeiss). Sections also were made after viral transfer for opsin verification, and these sections were stained with anti-rabbit GFP (1:500, #AB290, Abcam), CaMKII-α (6G9) mouse monoclonal antibody (mAb) (1:200, #50049, Cell Signaling Technology) antibodies, and cover-slipped with DAPI (1:200, Vector Laboratories). Secondary antibodies were anti-rabbit immunoglobulin G (IgG) conjugated to Alexa Fluor 488, and anti-mouse IgG conjugated to Alexa Fluor 647 (1:200, Life Technologies). Sections with RetroBeads solution (Lumafluor) injection were cover-slipped with DAPI (1:200, Vector Laboratories). Images were acquired with a Zeiss LSM 700 confocal microscope (Carl Zeiss).