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NeuroScience Associates

Fixation Methods and Perfusion Solutions


The best neurohistologic results are delivered when optimum procedures are followed for tissue preparation. Transcardial perfusion is the most effective fixation method and yields the best results.

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.
Prepare in the lab:

For 1000 ml TotalFor 2000 ml TotalFor 4000 ml TotalFinal %
Paraformaldehyde40 g80 g160 g4%
Phosphate Buffer
0.2M; pH 7.2
500 ml1000 ml2000 ml--
Note: Not to be used for the purpose of cryoprotection
40 g80 g160 g4%

For 1000 ml Total:

  1. Begin with 500 ml of dH20 and heat to 60°-65°C (~104° F) on a heater/ magnetic stirrer.
  2. Add the sucrose. Please note: Sucrose is optional and this solution is not to be used for cryoprotection.
  3. 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.
  4. Allow to cool to room temperature
  5. Bring the total volume to 1000 ml with 0.2M Phosphate Buffer and verify the pH.
  6. Filter the solution using a qualitative paper.

Protocol for making PBS buffer solution:


Stock Solutions:

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.

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.

Stock Dibasic Sodium Phosphate360ml720ml
Stock Monobasic Sodium Phosphate140ml280ml
Saline; NaCl4.5g9g


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

Spinal Cord Post-Perfusion

Brain Extraction

Brain Hemisection


Contact Technical Support at NSA Labs via this form or call 865.675.2245 for shipping and preparation questions.