4% paraformaldehyde in PBS

10/23/00

1. For 100mL. Best to use glassware, stir bar set aside for making paraformaldehyde only.

2. Put 50mL ddH2O in a glass beaker with a stir bar in the bottom. Put stirring on hot plate in the hood. Set hot plate to setting 3-4 (about 60 degrees, make sure the solution does NOT boil).

3. Measure out 4g of paraformaldehyde (Sigma, stored on the bottom left in the cold box). Measure IN THE HOOD (move the scale in there). Add the paraformaldehyde to the heater stirring water.

4. You need to raise the pH until the solution clears. I do this by adding a few drops (2-3) of 1N NaOH and watch the solution. Gabrielle does with 10uL of 10N NaOH and that is it. If the solution fails to clear, check the pH by pipetting a small amount of the solution onto a pH strip. If you are too high (>8) or too low (<7) keep pHing. You cannot pH on the pH meter as it will be ruined by the paraformaldehyde.

5. When dissolved, cool, then filter the solution by pouring through a piece of filter paper inside a funnel. Pour into a graduated cylinder to check the volume.

6. Add 10mL of 10X PBS. Add ddH2O to 100mL.

7. Recheck the pH with paper. Adjust carefully with small amounts of dilute HCl if needed.

8. Aliquot to use.