✓Perform vascular perfusion fixation through the heart using paraformaldehyde
✓Prepare surgical apparatus and materials for whole-animal perfusion
✓Dissect and preserve fixed brain tissue for downstream analysis
Protocol
Here we describe a low-cost, rapid, controlled and uniform fixation procedure using 4% paraformaldehyde perfused via the vascular system: through the heart of the rat to obtain the best possible preservation of the brain.
Difficulty
intermediate
Total time
~45 min per rat
Model organism
Rat
Biosafety
BSL-1
Steps
1
Prepare perfusion apparatus and materials
Assemble the perfusion rig, including peristaltic pump, tubing, and collection apparatus. Prepare 4% paraformaldehyde fixative and warm to appropriate temperature.
▶ 01:12
2
Perform perfusion surgery on rat
Anesthetize the rat, expose the heart via thoracotomy, cannulate the left ventricle, and sever the vena cava to allow outflow during perfusion.
▶ 02:58
3
Perfuse fixative through vascular system
Initiate perfusion pump to deliver 4% paraformaldehyde through the heart at controlled flow rate, ensuring uniform fixation of tissues including the brain.
▶ 05:08
4
Dissect and store fixed brain tissue
Remove the fixed brain from the skull and place in storage fixative or buffer. Document and store at appropriate temperature for downstream processing.
▶ 06:28
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