Oct 19, 2023

Public workspaceTranscardial Perfusion in Mouse

DOI
dx.doi.org/10.17504/protocols.io.dm6gpbrw1lzp/v1
  • Michael Henderson1
  • 1Van Andel Institute
Michael Henderson
Open access
DOI: dx.doi.org/10.17504/protocols.io.dm6gpbrw1lzp/v1
Protocol Citation: Michael Henderson 2023. Transcardial Perfusion in Mouse. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpbrw1lzp/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: January 04, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 56537
Keywords: Perfusion, heart, Transcardial Perfusion, Perfusion Solutions, ASAPCRN
Abstract
This protocol details about the transcardial perfusion in mouse.
Attachments
338-741.pdf
338-741.pdf
527KB
Materials
Materials Needed:

  • Aluminum-wrapped Styrofoam
  • Plastic lid (to capture runoff)
  • Labmat (on bench and on styrofoam)
  • Amount30 mL syringe with butterfly needle with cut tip
*Fill with 30 mL PBS and push PBS through tubing so all air bubbles are expelled.
  • Perfusion pump: 100-120 mL/hour

Dissection tools

  • 1 tube with 2 small drops heparin for blood
  • 1 tube for tail
  • 2 conicals with ethanol or formalin fixative
  • Amount30 mL /mouse Concentration0.1 Molarity (M) PBS + heparin (1:100)
  • 70% ethanol bottle

4% PFA in 0.1 M NaPO4 (1L)

Dissolve into 500ml diH20:
  • Amount11.36 g sodium phosphate dibasic
  • Amount2.76 g sodium phosphate monobasic
Once dissolved:
  • Add Amount40 g PFA (powder)
  • Fill to Amount1 L with diH20




Transcardial Perfusion
Transcardial Perfusion
Make mixture of ketamine:xylazine:acepromazine (4:2:1) sufficient for anesthesia of all mice (~Amount30 µL / Amount20 g mouse). Record ketamine used in controlled substance log book.
Apply anesthesia to one mouse via intraperitoneal injection, and place mouse in bucket long enough for anesthesia to take effect. Apply a hard toe pinch until mouse no longer reacts, ensuring that the mouse can no longer feel pain before proceeding.
Place mouse, abdomen-up, on Styrofoam block wrapped in lab mat. Spray mouse abdomen with 70% ethanol. Grasp skin below ribcage with forceps and cut skin with scissors from middle up either side towards the armpits, cutting through ribcage.
Note
Avoid blood vessels and organs. Diaphragm should carefully be cut circumferentially.

Remove pericardium and peripheral fat to expose heart. Hold back ribs with hemostats.
Note
* At this point blood can be taken via syringe from the right ventricle if desired. Amount200 µL to Amount300 µL should be sufficient.


Place blunted butterfly needle into left ventricle and cut right atrium. Push PBS through syringe by using perfusion pump at 2 mL/minute rate.
Note
Liver should lose pigment and effluent should be dark blood then become more PBS as perfusion continues.

After perfusion is complete, remove brain, spinal cord and other desired parts and transfer to fixative.
Note
* Brain can be removed by carefully drilling hole with scissors at olfactory bulb junction and spreading scissors apart so skull splits in half.



Perfusion Solutions
Perfusion Solutions
Weigh PFA under the hood and using bench pads.
Heat and stir (in chemical hood).
Lets dissolve the PFA around Temperature60 °C (use the thermometer). Do not let the temperature exceed Temperature65 °C (remove from hot plate temporarily if necessary).


Once dissolved but still foggy, Ph7.4 with NaOH (it should make the PFA clear).

Prepare a big filter funnel using Whatman filter paper.
Filter the 4% PFA into a liter bottle/w lid.
Store the PFA covered atTemperature4 °C .