Perfusion Solution

FOR BEST RESULTS WITH MOST STAINS, USE PERFUSION SOLUTION TYPE 3.

CONSULT WITH NSA PRIOR TO PERFUSION TO DETERMINE THE MOST APPROPRIATE SOLUTIONS TO USE.

Perfusion Solutions Type 3: ‘Superior Reagent Grade’

Na Cacodylate Based for NSA's Disintegrative Degeneration Stain

Pre-made NSA solutions are available from EM Sciences. To order, call (215) 412-8400.

Type 3 Perfusion Wash: catalog # 1222SK | Type 3 Perfusion Fix: catalog # 1223SK

Perfusion Wash:

  For 1000 ml Total For 2000 ml Total For 4000 ml Total Final %
NaCl 8g 16g 32g 0.8%
Dextrose 4g 8g 16g 0.4%
Sucrose 8g 16g 32g 0.8%
CaCl2 0.23g 0.46g 0.92g 0.023%
Sodium Cacodylate* 0.34g 0.68g 1.36g 0.034%

* e.g. Sigma Cat # C0250 Cacodylic acid, sodium salt OR call Black Scientific 508-650-1761

 

Perfusion Fix:

  For 1000 ml Total For 2000 ml Total For 4000 ml Total Final %
Paraformaldehyde 40 g 80 g 160 g 4%
Sucrose 40 g 80 g 160 g 4%
Sodium Cacodylate* 14.34 g 28.68 g 57.36 g 1.4%

 

For 1000 ml Total:

  1. Begin with 800ml dH2O of water and heat to 60°-65°C (~104° F) on a heater/ magnetic stirrer.

  2. Add the sucrose.

  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. Add the Sodium Cacodylate. The solution will clear considerably but still may be turbid and will require filtration with a qualitative paper.

  5. Allow to cool to room temperature.

  6. Bring the total volume to 1000 ml with dH2O. Adjust the pH to 7.2 - 7.4 with concentrated HCl dropwise (ca. 15-20 drops).

Preparation of tissue for disintegrative degeneration staining WHEN tissue is already in another fix:

For 1000 ml Total:

  1. Begin with 800 ml of dH2O. Add 14.34 g Sodium Cacodylate and dissolve thoroughly.

  2. Add 110 ml 37% commercial formaldehyde.

  3. Bring the total volume to 1000ml using dH2O.

  4. Adjust the pH to 7.2 - 7.4 with concentrated HCl dropwise (ca. 15-20 drops).

  5. Rinse the brains several times, 3-5 minutes each, in dH2O

  6. Place in above Perfusion Fix solution; change to a fresh solution after 2 weeks.

 

Sequence of Tissue Preparation Events with Links:

Fixation Method arrow Fix Solutions arrow Perfusion Instructions arrow Fix Timing & Storage Buffers arrow Shipping Tissue

 

Other Direct Links to Related Items:

Fixes:

Buffers:

Procedures:

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