Sep 20, 2022
Perfusion-fixation procedure for adult rhesus monkeys
- Yoland Smith1,2
- 1Emory University;
- 2ASAP
Protocol Citation: Yoland Smith 2022. Perfusion-fixation procedure for adult rhesus monkeys. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl8jn67g2w/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: September 14, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 70004
Keywords: Perfusion-fixation, Adult rhesus monkeys, ASAPCRN
Abstract
This protocol details Perfusion-fixation procedure for adult rhesus monkeys.
Attachments
527-1107.pdf
118KB
Materials
Solutions to prepare:
- Pentobarbital ( 25 mg/mL solution).
- Ringer’s solution
Dissolve the following reagents, in order, in 1000 mL of distilled water.
| A | B | |
| Reagent | Quantity | |
| HEPES | 0.60 g | |
| NaCl | 11.86 g | |
| KCl | 0.223 g | |
| CaCl2 | 0.353 g | |
| NaHCO3 | 2.18 g | |
| KH2PO4 | 0.177 g | |
| MgSO4 | 0.32 g | |
| D-glucose | 1.8 g |
- Fixative 2.5 L
- Paraformaldehyde 96%Fisher ScientificCatalog #AC416785000 )
2. 0.1% GlutaraldehydeElectron Microscopy SciencesCatalog #16220
3. 0.1 Molarity (M) phosphate buffer, 7.4
Note
Glutaraldehyde must be added to the fixative solution only at the time of perfusion.
FIXATIVE SOLUTION:
Safety information
PREPARE THIS SOLUTION UNDER A HOOD.
- Final volume: 1 L
- Concentration:
| A | B | |
| Paraformaldehyde | 4% | |
| Glutaraldehyde | 0.1% |
- Heat 500 mL distilled water until the temperature reaches 60 °C .
- Dissolve 40 g paraformaldehyde with strong stirring action for 00:20:00 .
- Add NaOH drop by drop until the solution is almost clear.
- Let the solution cool for 01:00:00 .
- Filter the solution in another beaker and add 500 mL PB (0.2 Molarity (M) 7.4 )
Note
KEEP IN THE FRIDGE.
- Just before the perfusion, substitute 4 mL of fixative by 4 mL of glutaraldehyde 25%.
ANTI-FREEZE SOLUTION:
Prepare the following solution in the same order:
For 1000 mL solution:
| A | B | |
| Sodium phosphate monobasic, NaH2PO4·H2O | 13.8 g | |
| Sodium phosphate dibasic heptahydrate, Na2HPO4·7H2O | 25.8 g | |
| Distilled water | 400 ml | |
| Ethylene glycol | 300 ml | |
| Glycerol | 300 ml |
Note
Sections can be stored in this solution at -20 °C freezer for many months without loss of ultrastructural preservation and tissue antigenicity.
Equipment and room needed:
- Necropsy room equipped with proper exhaust and ventilation to reduce exposure to aldehydes.
Equipment
General-Purpose Peristaltic Pumps
NAME
Peristaltic Pump
TYPE
Fisherbrand™ GP1000
BRAND
13310656
SKU
LINK
.
- Oxygen tank (95% O2, 5%CO2).
- Surgical instruments (scalpels, hemostats of various sizes, bone cutter, scissors, bone saw, forceps, rongeur).
Before start
Note
Wear PPE before entering the perfusion room, i.e., gloves, coat, hair net, safety glasses, face mask and face shield or goggles.
Safety information
Make sure the Hazardous Waste container is not full. Replace if full.
Perfusion-fixation procedure for adult rhesus monkeys
Perfusion-fixation procedure for adult rhesus monkeys
1d
1d
Add glutaraldehyde to the fixative solution.
Start oxygenation of the Ringer’s solution.
Anesthetize the animal with Ketamine (10 mg/kg , i.m.) or Telazol (3 mg/kg -5 mg/kg , i.m.) in its home cage and bring to the necropsy room.
Place an i.v. catheter.
Perform a tracheal intubation.
Inject 1 mL of heparin (i.v.).
Inject an overdose of pentobarbital (25 mg/kg i.v.).
Ensure the absence of all reflexes, including toe pinch reflexes and brainstem reflexes, such as corneal reflexes.
Cut the thoracic cage to provide access to the heart, clamp the descending aorta, open the pericardium and expose the heart muscle, make an incision of the right atrium and insert the perfusion needle in the left ventricle.
Artificially ventilate the animal through the tracheal tube during the Ringer’s solution perfusion.
Infuse transcardially ~300-400 mL of cold oxygenated Ringer’s solution at a rate of 80-90 ml/min through a needle (14G; 1.5 inch long) inserted in the left ventricle. Let the infused solution exit the vascular system through a hole in the right atrium.
Perfuse 2.5 L of fixative, starting at a rate of 80-90 ml/min for the first liter and then reduce the rate to 50 ml/min for the remaining solution.
After perfusion, fix the animal’s head in a stereotaxic frame and open the skull using a bone saw and rongeur.
Cut the brain in 10 mm -thick blocks in the coronal stereotaxic plane, remove the resulting blocks of tissue from the skull and post-fix them for 24:00:00 in 4% paraformaldehyde solution in PB (0.1 Molarity (M) , 7.4 ) at 40 °C .
1d
Transfer the tissue into a phosphate-buffered saline (PBS, 0.01 Molarity (M) , 7.4 ) solution.
Cut the brain in 50 µm -thick sections using a vibrating microtome or a freezing microtome and store in a -20 °C freezer in an anti-freeze solution ( Please refer material section) until further processing.