G. Buffer recipes

A guideline procedure and tips for staining of paraffin embedded sections, including antigen retrieval, chromogenic detection and fluorescent detection

Fixation:

Formalin Solution (10%, buffered neutral):

Formaldehyde (37-40%) 100 ml
Distilled water 900 ml
NaH2PO4 4.0 g
Na2HPO4 (anhydrous) 6.5 g

Mix to dissolve.

Paraformaldehyde (4%, buffered neutral):

8% Paraformaldehyde:
Paraformaldehyde 40 g
Distilled water 500 ml

0.2M Phosphate Buffer (PB), pH 7.4:
Na2HPO4 10.9 g
NaH2PO4 3.2 g
Distilled water 500 ml

The solution should be at pH 7.4. Do not pH using acid or base! If you need to adjust the pH, make up a separate 0.2M solution of either the monobasic or dibasic sodium phosphate (depending on how you need to adjust the pH) and add accordingly.

Heat 8% PFA solution at 60ºC while stirring (do not allow the solution to go above 60ºC). Once the solution has reached 60ºC and the PFA is dissolved, add 500 ml of 0.2M phosphate buffer, to bring the solution to 4% PFA in 0.1M phosphate. Carefully add 1N NaOH dropwise until solution is clear (try 1-2 drops per 500 ml; if still not clear, add a few more drops. Alternatively, you can add 2 pellets of solid NaOH in 1-2 L of solution). Cool the solution and filter.

Top tip Molly

PFA should always be made up fresh on the same day you wish to use it. Storage overnight at 4oC is possible, but it will not fix as well the second day. It is possible possible to freeze the PFA solution at -20°C, but for consistency of results, tissue should either be fixed always with fresh PFA or always with freshly thawed PFA.

Bouin Solution (especially for preserving soft and delicate structures such as brain tissues)

Picric acid (saturated) 75 ml
Formaldehyde (37-40%) 25 ml
Glacial acetic acid 5 ml

Mix well.

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