Dec 13, 2024
Version 2
Mouse Perfusion Protocol V.2
- 1Cornell University
Protocol Citation: Heath Robinson 2024. Mouse Perfusion Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l29qr3v1y/v2Version created by Heath Robinson
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: December 11, 2024
Last Modified: December 13, 2024
Protocol Integer ID: 115533
Keywords: Perfusion
Funders Acknowledgements:
Heath Robinson
Grant ID: F32MH134673
Disclaimer
DISCLAIMER – FOR INFORMATIONAL PURPOSES ONLY; USE AT YOUR OWN RISK
The protocol content here is for informational purposes only and does not constitute legal, medical, clinical, or safety advice, or otherwise; content added to protocols.io is not peer reviewed and may not have undergone a formal approval of any kind. Information presented in this protocol should not substitute for independent professional judgment, advice, diagnosis, or treatment. Any action you take or refrain from taking using or relying upon the information presented here is strictly at your own risk. You agree that neither the Company nor any of the authors, contributors, administrators, or anyone else associated with protocols.io, can be held responsible for your use of the information contained in or linked to this protocol or any of our Sites/Apps and Services.
Abstract
Protocol for perfusing mice with fixative, including description of creating all required solutions.
Materials
Equipment:
Surgical Platform
Surgical platform at an angle that allows for flow of fluids off platform waste disposal.
Equipment
Surflo winged infusion set 21G 3/4
NAME
Terumo
BRAND
TER 3SV-21BLK
SKU
Equipment
Insulin gauge needle (BD Ultra-fineTM II, NJ, USA)
NAME
(8 mm x 0.3 mm)
TYPE
BD
BRAND
328838
SKU
LINK
0.3 mL
SPECIFICATIONS
Equipment
Variable-Flow Peristaltic Pump
NAME
Fisherbrand
BRAND
13-876-2
SKU
https://www.fishersci.com/shop/products/fisher-scientific-variable-flow-peristaltic-pumps-4/p-158215
LINK
Reagants:
PBS Tablets, Phosphate Buffered Saline, Fisher BioReagentsFisher ScientificCatalog #BP2944100
Paraformaldehyde Powder (PFA)Contributed by usersCatalog #P6148
Sodium HydroxideFisher ScientificCatalog #BP359500
1000ml of 4% PFA solution preparation:
1. Measure 600 mL of ddH2O in
a graduated cylinder, and heat it to 70 °C to 90 °C (note: not directly heating with
buffer).
2. Weigh 40 g of Paraformaldehyde Powder (PFA)Contributed by usersCatalog #P6148 , and dissolve it in the above-mentioned solution that is continuously heated, stir, and add an appropriate amount of NaOH (about 0.1 g for 1000 mL ) to aid the PFA dissolving.
3. Add 10 tablets of PBS Tablets, Phosphate Buffered Saline, Fisher BioReagentsFisher ScientificCatalog #BP2944100
4. Make the volume to 1000 mL .
5. Adjust the PH value to
7.2 -7.4 (<0.3 g Sodium HydroxideFisher ScientificCatalog #BP359500 , need not adjust the PH).
6. Filter with
Equipment
Millipore® Stericup® Quick Release Vacuum Filtration System
NAME
0.22 um polyethersulfone membrane filtration system
TYPE
Millipore
BRAND
All Photos(3) Key Documents COA/COQ View All Docum
SKU
LINK
0.22 um filter
SPECIFICATIONS
7. Bottle and store at 4 °C until use.
Safety warnings
Paraformaldehyde (PFA) and sodium hydroxide are hazardous substances that can be dangerous if not handled safely. Waste includes previous hazardous chemicals, as well as potential biological hazards. Handling should only be done with proper peronsal prote
Ethics statement
Experiments involving animals must be conducted according to internationally accepted standards. Users should always have prior approval from an Institutional Animal Care and Use Committee (IACUC) or equivalent ethics committee(s). Prior ethics approval should be obtained before performing these experiments.
Before start
Ensure the animal is under proper anesthesia plane per local University and IACUC standards.
Perfusion Steps
Perfusion Steps
Rinse the peristaltic pump thoroughly with saline or PBS for more than 5 minutes
Start the peristaltic pump with normal saline/PBS, make sure everything is well
connected (pump, tube, needles, saline/PFA), and there are no air bubbles in
the tube.
After anesthesia, the animals are fixed tightly with pins on four paws. Open the abdominal
and thoracic cavity with forceps in the left hand and scissors in the right hand. Remember that the tips of scissors are as close as possible to the bone to reduce the possibility of cutting the blood vessels.
Safety information
Anesthesia for euthanasia must follow and abide by local IACUC and University standards. Ensure proper anesthesia plane before performing perfusion.
Cut a small hole in the right atrium, inject saline with a needle connected to a tube in the pump into the left ventricular, and you will find blood flowing out of the right atrium.
Note
The angle of injection should be 45 degrees towards the Aortic Arch
Perfuse with PBS for less than 5 minutes and stop the pump when the
saline running out of the right atrium is clear (no blood is found). Change the tube from
a saline bottle to a 4% PFA bottle (stop the pump first and change the tube second to
reduce the possibility of bubbles in the tube). Open the pump again and perfuse
animals with 4% PFA for another 10 minutes.
Note
Optimal flow settings for listed Peristaltic Pump (below) is between 32-25.
Equipment
Variable-Flow Peristaltic Pump
NAME
Fisherbrand
BRAND
13-876-2
SKU
https://www.fishersci.com/shop/products/fisher-scientific-variable-flow-peristaltic-pumps-4/p-158215
LINK
Note
Proper perfusion will result in tail rising upwards upon PFA perfusion.
Safety information
Paraformaldehyde (PFA) is a hazardous substance that can be dangerous if not handled safely
10 minutes for adults and 7 minutes for younger than P10.
Dissect the brain, and keep the tips of scissors as close as possible to the bone. This
will reduce the damage to the brain tissue. Post fixed tissues in 4% PFA for
less than 1 hour or in PFA overnight.
Safety information
Dispose of waste in accordance with local EHS standards.