pH-Shift/Formaldehyde Fixation

This protocol was used for staining of Glogi apparatus with lectins and nuclei.

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. Cells are washed with phosphate-buffered saline to remove medium and serum proteins.
3. Cells are fixed 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. Dilute fluorescently-labeled wheat germ agglutinin (WGA) to 10 ug/mL in PBS. Transfer the coverslips (cell side up) to a piece of Parafilm in an airtight humidified chamber. Apply enough diluted WGA to cover the entire coverslip and incubate for 1 hour. For labeling DNA, dilute DAPI to 10 ug/mL into PBST (from a 1 mg/mL stock in water) and stain for 20 minutes.
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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