Sample Fixation Protocols

pH-Shift/Formaldehyde Fixation

1. Plate cells on acid-washed coverslips and allow to grow to 60 - 70% confluence. Coverslips are cleaned by soaking in nitric acid overnight and thoroughly rinsed with de-ionized water.
2. Wash cells with phosphate-buffered saline to remove medium and serum proteins.
3. Fix cells with 3% paraformaldehyde in PEM buffer (0.1 M PIPES, pH 6.5; 1 mM MgCl2; 1 mM EGTA) for 5 minutes at room temperature. Aspirate the fixative and replace with 3% paraformaldehyde in borate buffer (0.1 M sodium borate, pH 11; 1 mM MgCl2) for 10 minutes.
4. Permeabilize the cell membranes with a solution of 1% Triton X-100 in PBS for 15 minutes.
5. Remove detergent with 3 rinses with PBS/pH 8.
6. Reduce unreacted aldehydes in the sample by treatment for 10 minutes with a solution of 1 mg/mL sodium borohydride dissolved in PBS/pH 8 immediately before use. Repeat one more time.
7. Wash the cells with 3 brief rinses with a solution of 0.1% Triton X-100 in PBS (PBST).
8. If antibodies are to be used, dilute them to their appropriate concentration in 5% normal goat serum in PBST. Transfer the coverslips (cell side up) to a piece of Parafilm in an airtight humidified chamber. Apply enough diluted primary antibody to cover the entire coverslip and incubate for 1 to 2 hours..
9. Wash 3 times, briefly, with PBS (no detergent) and one time with de-ionized water.
10. Mount coverslips (cell side down) in an appropriate mounting medium. It is often desirable to include anti-fading agents to decrease photobleaching during observation. There are several excellent commercially-available mounting media, the best of which (in our experience) is ProLong from Molecular Probes. Alternatively, a solution of 90% glycerol 10% sodium borate (from 1 mM aqueous solution, pH 9) supplemented with 3% n-propyl gallate works well. Mount the coverslip in a drop of this, aspirate off the excess from around the edges of the coverslip, and seal the edges with clear nail polish.

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