4% PFA




Final Concentration


MQ water


1500 ml



PBS (20x)


100 ml


Paraformaldehyde (Sigma,


80 g




18 pellets


Adjust the pH to 7.4 and then the volume to 2000 ml with MQ water. Keep refrigerated.


  1. Heat up the 1x PBS buffer in the microwave for 6 min (temperature will be about 64C). In the fume hood keep the solution on hot plate while stirring and add the NaOH pellets. It takes about 5 min to dissolve completely. Take care not to boil the solution (keep it at 64-54 c).
  2. Add 80 g of paraformaldehyde powder. Stir until dissolved, it takes about 15 min.
  3. Once the paraformaldehyde is dissolved, the solution should be cooled (and filtered).
  4. Adjust the pH to 7.4 with HCl (5M) and then the volume to 2 L with MQ water.
  5. The solution can be aliquoted and frozen or stored at 2-8 °C for up to one month. But we prefer freshly made and used within 4 days.