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NeuroScience Associates

Post-Fixation Buffer Solutions, Storage and Timing

 

Recommended time to remain in fix after perfusion is 12-16 hours.

  • Antibody staining: for formalin sensitive antibody staining 4 hours post perfusion is sufficient

Following fixation, transfer tissue into Phosphate-Buffered Saline (PBS) storage buffer, which is acceptable for all stains.

  • Amino CuAg (degeneration staining): wait at least 24 hours before extracting brain to allow tissue to fully harden, preventing the induction of artifacts during staining

 

Protocol for making PBS buffer solution:


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.

 500ml1000ml
Monobasic Sodium Phosphate140ml280ml
Dibasic Sodium Phosphate360ml720ml
Saline; NaCl4.5g9g
pH7.27.2

 

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).

 

Also see: Use of Cryroprotectant to Maintain Longterm Peptide Immunoreactivity and Tissue Morphology by Hoffman and Le.

 

For more information:

Antigen Preserve (AP) Solution

Mounting Solutions

Perfusion Fix Solutions

Fixation Methods & Perfusion Solutions

Perfusion Protocol, Pump Calibration

Spinal Cord Post-Perfusion

Brain Extraction

Brain Hemisection

 

Consult with NSA to confirm the appropriate buffer and fixation time for your study via this form or call 865.675.2245.