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Paraformaldehyde Fixative

For 500 ml 2% Paraformaldehyde solution:

1. Allow paraformaldehyde (PFA) powder (Aldrich #30525-89-4 or Sigma P-6148) to come to room temperature (Stored in refrigerator)
2. Weigh 10.0 g PFA in fume hood
3. Flush container with Argon or Nitrogen to prevent air decomposition of p-formaldehyde.
4. Dissolve in 475 ml distilled H₂O in 60-70º C water bath on hot plate in fume hood. Do not allow water bath to go over 70ºC (formaldehyde will vaporize!).
5. While disolving, label 100 X 4 ml and 7 X 12 ml tubes (or other combinations of useful aliquots) with:

   2% PFA and the date

6. After @ 1 hr add 1 or 2 drops of 5M NaOH. Cloudy suspension will then turn clear.
7. Allow to cool to room temperature (@ 2 hours).
8. Add 25 ml 20X PBS (see recipe) and adjust pH to 7.3.
9. Filter, aliquot into tubes and freeze. Aliquots good for at least 5 years.

Working Diltution:

Thawed aliquots are stable at 4º C for up to 2 weeks.

Dilute 1 part 2% PFA to 3 parts cells in PBS (eg 60 µl + 180 µl to yield 0.5% final concentration).

References:

1. Becton Dickinson Immunocytometry Systems Source Book (1989) 2.10
2. Lanier, L.L., and Warner, N.L. (1981) Paraformaldehyde Fixation of Hematopoietic Cells for Quantitative Flow Cytometry (FACS) Analysis. Journal of Immunological Methods 47, 25

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Dennis J. Young, UCSD Cancer Center Flow Cytometry Core Facility