Fixation of Cells with 4% Paraformaldehyde
The following protocol provides information for the preparation of 3x PBS and 4% paraformaeldehyde, which is used as a cell fixative.
Preparation of 3x PBS
1. Prepare two separate solutions composed of:
390 mM NaCl, 30 mM Na2HPO4
390 mM NaCl, 30 mM NaH2PO4.
2. Mix to obtain 3x PBS, pH 7.2
3. Either autoclave or filter to sterilize.
Preparation of 4% Paraformaldehyde
1. To prepare 30ml of 4% paraformaldehyde:
2. Weigh out 1.2g of paraformaldehyde
3. Add 19ml of H2O and heat to 60oC
4. In a fume hood, add 10ul of 5M NaOH. If necessary, increase the volume of NaOH added to the solution until the paraformaldehyde is dissolved [note – some particles of paraformaldehyde may not fully dissolve].
5. Add 10ml of 3x PBS.
6. Bring the pH of the solution to 7.2 by adding small aliquots (approx 5ul) of 5M HCl.
7. Make final volume to 30ml with H2O.
8. Filter solution through a 0.45micron filter.
9. Cool on ice to 4oC prior to use.
CAUTION – Paraformaldehyde is a carcinogen!
Fixation of Cells
1. Remove growth media from cells.
2. Add sufficient 4% paraformaldehyde to generously cover the cell monolayer.
3. Incubate at 4oC for approx. 30 min to complete cell fixation.
4. Cells can be stored at 4oC in paraformaldehyde for several days before use.