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Making 4% para-formaldehyde

1. Weigh out enough powder or prill form para-formaldehyde to make a 4% solution.

2. Add ddH20 to approximately 50-75% of total volume to be used.

3. Heat carefully to 55-60*C (solution will be cloudy white) and start adding 1,5, or 10M NaOH (the larger the volume the higher the concentration of NaOH you may want to use) until solution clears. Make sure solution does not rise above 65*C. If it does, start over!

4. Filter clear solution with filter paper and a vaccum system to remove large impurities (this may sound trivial but it is essential).

5. Add appropriate amount of 10X PBS or 10X buffer of choice, then bring up to final volume. (Make sure there are no amines in the buffer, i.e. Tris-Glycine etc.)

6. pH solution to approximately 7.4 or slightly higher (I routinely use a pH of ~7.6; para-formaldehyde fixes better at higher pH's).

7. Aliquot into 50 or 15ml tubes and store at -20*C, thaw when needed. Thaw at room temperature, you can thaw at 37*C if you're in a hurry, but NEVER at 50*C or higher.
You can refreeze and thaw up to 3X's (that's the most I've personally refrozen an aliquot; you can probably do it several times more.

8. After you thaw the aliquot, make sure it is mixed well by shaking it several times (You may initially get a phase effect like when you pour ddH20 into a highly concentrated solution).