Preparation Of Fixation Solutions

Selection of a fixative is very critical to the preservation of cellular or tissue morphology during in situ hybridization. Based on our experience, 10% buffered-for-malin solution (pH 7.0) is an excellent fixative, or a paraformaldehyde solution can be used as a fixative as prepared below.

Preparation of 4% (w/v) Paraformaldehyde (PFA) Fixative (1 l)

1. Carefully weigh out 40 g paraformaldehyde powder and add it to 600 ml preheated dd.H2O (55 to 60°C) and allow paraformaldehyde to dissolve at the same temperature with stirring.

Caution: Paraformaldehyde is toxic and should be handled carefully. The preparation should be carried out in a fume hood on a heating plate. Temperature should not be above 65 °C and the fixative should not be overheated.

2. Add one drop of 2 N NaOH solution to clear the fixative.

3. Remove from heat and add 333.3 ml (1/3 total volume) of 3X PBS (8.1 mM KCl, 4.5 mM KH2PO4, 411 mM NaCl, 45 mM Na2HPO4, pH 7.2, autoclaved).

4. Adjust pH to 7.2 with 4 N HCl and bring final volume to 1 l with dd.H2O.

5. Filter the solution to remove undissolved fine particles, cool to room temperature and store at 4°C until use.

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