Paraformaldehyde Fixation

From LPDwiki

About this protocol: This protocol outlines the steps and materials involved in fixing cells using 4% paraformaldehyde in PBS. Use this protocol for preparing cells for immunostaining.

Necessary Materials

1. Phosphate Buffered Saline without Ca and Mg (PBS[-])

  • Purchased from Corning (21-040-CV) and should be stored at 4°C.

2. 4% Paraformaldehyde in PBS (PFA)

  • Purchased from Affymetrix (19943 1 LT) and should be at 4°C upon arrival in a flammable safe refrigerator.
  • Note: Paraformaldehyde is a carcinogen and should only be used under an appropriate solvent fume hood.

Procedure

Solution Preparation

Appropriate amounts of PBS[-] and PFA will depend on the number of samples you will fix

1. Individually aliquot PBS[-] and PFA into conical tubes.

  • Note: PFA must be handled under a fume hood!

2. Warm up the aliquots of PBS[-] and PFA to 37°C in the water bath.

Fixation Protocol

This protocol should be done in a lab space that has a solvent fume hood!

1. Using 1000 uL pipettes, remove the cell culture media from each well.

2. Replace contents of each well with warmed PBS[-] and let sit for 1 minute.

PBS[-] volume per well size:
24 well plate 12 well plate 6 well plate
500 uL 1 mL 2 mL

3. Repeat washing with PBS[-] for two (2) additional times.

4. Remove PBS[-] and replace with warmed 4% PFA solution.

  • Note: Must be done in fume hood!
4% PFA volume per well size:
24 well plate 12 well plate 6 well plate
500 uL 1 mL 2 mL

5. Incubate samples with 4% PFA solution for 20-25 minutes.

  • Note: Must be done in fume hood!

6. Remove 4% PFA from samples and place in a separate waste container labeled "PFA Waste".

  • Note: Must be done in fume hood!

7. Replace contents of each well with PBS[-] and let sit for 1 minute.

8. Remove PBS[-] from first wash and place with the PFA in the "PFA Waste" container.

9. Wash two (2) additional times with PBS[-].

10. Fill up wells with PBS[-] and seal with Parafilm.

11. Label with date fixed and time of fixation. Store at 4°C.

Cleaning Up

1. Dispose of non PFA contaminated waste (cell culture media/PBS[-]) by rinsing down the drain.

2. Dispose of PFA Waste in a designated hazardous chemical disposal area.

3. Dispose of used conical tubes and pipettes into the biological waste bin.