Post-Fixation Buffer Solutions, Storage and Timing
Recommended time to remain in fix after perfusion is 16-24 hours.
Following perfusion fixation and overnight in the same fixative solution (∼40ml with mild agitation), 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 which will prevent the induction of staining artifacts
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).
Also see: Use of Cryroprotectant to Maintain Longterm Peptide Immunoreactivity and Tissue Morphology by Hoffman and Le.
For more information:
Antigen Preserve (AP) Solution
Fixation Methods & Perfusion Solutions
Perfusion Protocol, Pump Calibration
Consult with NSA to confirm the appropriate buffer and fixation time for your study via this form or call 865.675.2245.
