Dissection and immunohistochemistry of mouse brainstem
Seol-Hee Kim, Thomas Taylor-Clark
Abstract
Mice are euthanized, perfused with fixative for brainstem tissue collection. Mouse brainstem is cryosectioned and slices are stained for protein expression using immunohistochemistry. Expression of specific proteins and reporter proteins are visualized using microscopy.
Steps
Tissue collection and cryosection
Mice are euthanized by CO2inhalation and transcardially perfused with ice-cold PBS followed by perfusion fixation with ice-cold 3.7% formaldehyde (FA).
Brainstem is dissected out and post-fixed for 4 h in 3.7% FA at 4°C. The brainstem is washed in PBS to remove residual FA and transferred to 30% sucrose solution for cryoprotection.
The brainstem is mounted in OCT (optimal cutting temperature) compound and snap frozen in dry ice. The brainstem is sectioned in 40 μm slices.
All slices are collected onto SuperFrost Plus slides. Slides are then air dried at room temperature in the dark overnight.
Immunohistochemistry
Tissue are permeabilized with 0.3% Triton X-100 in PBS (PBSTx) for 15 min.
Tissue are blocked with 1% bovine serum albumin (BSA)/10% donkey serum (DS)/0.3% PBSTx. for 1 h at room temperature.
Tissue are incubated with primary antibodies diluted in blocking buffer overnight at 4°C.
Tissue are washed with 0.2% Tween 20 in PBS (PBST) three times for 10 min.
Tissue are incubated with secondary antibodies in 1% BSA/5% DS in 0.2% PBST for 1 h at room temperature.
Tissue are washed with 0.2% PBST three times for 10 min and rinsed briefly with H2O.
Slides are air-dried and mounted with DPX Mounting Medium (Sigma-Aldrich) for imaging.
