Fixation Methods and Perfusion Solutions
For some studies, perfusion is not possible. Immersion fixation or “drop fix” is acceptable except for the Amino Cupric Silver Stain (degeneration stain), because red blood cells will stain. If the animal can be perfused with buffer and then placed into fix, this would be suitable for all stains, including this degeneration stain.
Perfusion Fix Solutions
High quality perfusion fix solutions yield high quality results. Commercially available formaldehyde solutions are NOT fresh and usually have added ingredients to stabilize the solution for long shelf life. This should be considered a “technical grade” perfusion fix solution. Such solutions should only be used for standard, routine, classical stains. However, even these stains may be optimized by having a freshly prepared perfusion solution using reagent grade components.
Preferred Perfusion Fix Solution
Phosphate-Buffered Paraformaldehyde:
Order from EM Sciences, call (215) 412-8400; catalog # 1224SK.
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Prepare in the lab:
| For 1000 ml Total | For 2000 ml Total | For 4000 ml Total | Final % | |
|---|---|---|---|---|
| Paraformaldehyde | 40 g | 80 g | 160 g | 4% |
| Phosphate Buffer 0.2M; pH 7.2 | 500 ml | 1000 ml | 2000 ml | -- |
| Sucrose Note: Not to be used for the purpose of cryoprotection | 40 g | 80 g | 160 g | 4% |
For 1000 ml Total:
- Begin with 500 ml of dH20 and heat to 60°-65°C (~104° F) on a heater/ magnetic stirrer.
- Add the sucrose. Please note: Sucrose is optional and this solution is not to be used for cryoprotection.
- Create a deep vortex using a magnetic stirrer bar. Gradually sprinkle the finely powdered paraformaldehyde onto the vortex surface. The solution will be quite milky.
- Allow to cool to room temperature
- Bring the total volume to 1000 ml with 0.2M Phosphate Buffer and verify the pH.
- Filter the solution using a qualitative paper.
Protocol for making PBS buffer solution:
Stock Solutions:
Dibasic
Na2HPO4 • 2H2O, M. wt. 178.05; 0.2M -solution contains 35.61 g/l.
Na2HPO4 • 12H2O, M. wt. 358.22; 0.2M -solution contains 71.64 g/l.
Monobasic
NaH2PO4 • H2O, M. wt. 138.01; 0.2M-solution contains 27.6 g/l.
NaH2PO4 • 2H2O, M. wt. 156.03; 0.2M-solution contains 31.21 g/l.
Depending on how many hydrogens are on each of the sodium phosphates you have to pick the one you have from the two choices for each of the monobasic and dibasic.
Make a stock solution for each by mixing the number of grams per liter as shown above.
| 500ml | 1000ml | |
|---|---|---|
| Stock Dibasic Sodium Phosphate | 360ml | 720ml |
| Stock Monobasic Sodium Phosphate | 140ml | 280ml |
| Saline; NaCl | 4.5g | 9g |
| pH | 7.2 | 7.2 |
Concentrated PBS is also commercially available from Sigma-Aldrich, Catalog# 79383-1L.
Phosphate-Buffered Saline (PBS) with 0.01% sodium azide should be used for tissue stored over three (3) weeks. Storage for this time period should take place in a refrigerator (4 degrees C).
For more information:
Perfusion Protocol, Pump Calibration
Post-Fixation Buffer Solutions, Storage, Timing
Contact Technical Support at NSA Labs via this form or call 865.675.2245 for shipping and preparation questions.