Aug 22, 2022

Public workspaceTranscardial perfusion of mouse tissues

DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzywnxlx1/v1
  • Rain Kwan1,
  • Courtney Wright1,
  • Louise Cottle1,
  • Alejandra Rangel1,
  • Asheeta Prasad1
  • 1The University of Sydney
Benjamin Trist
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzywnxlx1/v1
Protocol Citation: Rain Kwan, Courtney Wright, Louise Cottle, Alejandra Rangel, Asheeta Prasad 2022. Transcardial perfusion of mouse tissues. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzywnxlx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68574
Keywords: Paraformaldehyde, Perfusion, Mouse, Fixation, ASAPCRN, transcardial perfusion of mouse tissue, transcardial perfusion, fixation of mouse brain tissue, mouse tissue, mouse brain tissue, mice with sodium pentobarbitone, clear blood, preparation for immunohistochemical staining, saline to clear blood, immunohistochemical staining, preparation, sodium pentobarbitone, tissue, mice
Funders Acknowledgements:
Michael J Fox Foundation
Grant ID: ASAP-000497
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.
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Materials
Equipment:

  • Peristaltic pump, tubing and gavage
  • Surgical tools – haemostatic forceps, scissors, pins

Consumables:

  • Amount5 mL , Amount50 mL sample containers
  • Amount1 mL syringe

Key reagents:

  • Sodium pentobarbitone
  • Isopentane
  • Paraformaldehye (PFA)

Solutions:

  • 10x PBS
1. Amount77.3 g of NaH2PO4.H2O (Concentration0.28 Molarity (M) ), Amount203.7 g of Na2HPO4 (Concentration0.72 Molarity (M) ), Amount177.4 g of NaCl (Concentration1.5 Molarity (M) ) in Amount2 L dH2O, pH 6.9
  • 1x PBS, pH 7.4
1. Amount100 mL of 10x PBS in Amount900 mL dH2O, no pH adjustment required
  • 4% PFA in 1x PBS pH 7.4
1. See related protocol – Preparation of 4% paraformaldehyde solution for transcardial perfusions and histology.

Material input

Living athymic mice grafted with Day 25-35 human iPSC-derived neural progenitor cells.
Troubleshooting
Experimental Outline
1d 0h 16m
Attach a needle into the peristaltic pump tubing and prime the tubing by filling with TemperatureRoom temperature 1x PBS.
Lethally overdose mice with sodium pentobarbitone (Amount100 mg/kg ) via intraperitoneal injection.
Note
Anaesthetic depth is confirmed by the absence of withdrawal reflex, lack of response to both toe and tail pinch and a low respiratory rate.

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.
Note
These may prevent full occlusion of the vessel. Gently move the lungs with a micro spatula to uncover the descending aorta.

Insert the gavage needle attached to the pump tubing into the apex of the heart, advancing Thikness2 mm -Thikness3 mm into the tissue.

Clamp the heart and enveloped needle with forceps.
Note
Ensure the tip of the needle is not occluded by clamped forceps.
Cut the right atrium and turn on the perfusion pump at a rate of approximately 5mL/minute.
Perfuse the tissues for Duration00:05:00 with TemperatureRoom temperature 1x PBS.

5m
Crimp the tubing to prevent air bubbles entering the line and then switch the source solution for the perfusion pump from TemperatureRoom temperature 1x PBS to ice cold 4% PFA in 1x PBS.
Re-start the perfusion pump at 5mL/minute and perfuse the tissues for a further Duration00:08:00 .
Note
Fixation tremors should be observed after Duration00:01:00 -Duration00:02:00 .

8m
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 Temperature4 °C for up to Duration24:00:00 .
1d
Discard PFA solution into appropriate waste disposal stream.