Transcardial perfusion of mouse tissues
Alejandra Rangel, Rain Kwan, Courtney Wright, Asheeta Prasad, Louise Cottle
Abstract
This protocol describes how to perform transcardial perfusion and fixation of mouse brain tissues in preparation for immunohistochemical staining or histology. This process includes lethal overdose of mice with sodium pentobarbitone, occlusion of the descending aorta, followed by transcardial perfusion with phosphate buffered saline to clear blood from the vasculature and paraformaldehyde to fix mouse brain tissues.
Attachments
Steps
Experimental Outline
Attach a needle into the peristaltic pump tubing and prime the tubing by filling with Room temperature 1x PBS.
Lethally overdose mice with sodium pentobarbitone (100mg/kg) via intraperitoneal injection.
Working in a fume hood, make a lateral incision just beneath the rib cage, immediately inferior to the xyphoid process.
Cut through the diaphragm to expose the thoracic cavity.
Cut through the rib cage on the lateral edge, and then reflect the sternum above the head and hold in place with a pair of forceps.
Complete occlude the descending aorta using a clamp lacking teeth.
Insert the gavage needle attached to the pump tubing into the apex of the heart, advancing 2 mm -3 mm into the tissue.
Clamp the heart and enveloped needle with forceps.
Cut the right atrium and turn on the perfusion pump at a rate of approximately 5mL/minute.
Perfuse the tissues for 0h 5m 0s with Room temperature 1x PBS.
Crimp the tubing to prevent air bubbles entering the line and then switch the source solution for the perfusion pump from Room temperature1x PBS to ice cold 4% PFA in 1x PBS.
Re-start the perfusion pump at 5mL/minute and perfuse the tissues for a further 0h 8m 0s.
Collect and store PFA run-off for disposal.
Excise the brain and place immediately in a 50 mL sample container filled with ice-cold 4% PFA in 1x PBS. Place sample container on vertical rocker at 4°C for up to 24h 0m 0s.
Discard PFA solution into appropriate waste disposal stream.
