Sample Fixation Protocols

Below, I have provided example protocols for immunofluorescence staining of cultured cells. These protocols were adapted from Bacallao and Steltzer² specifically to preserve cellular three-dimensional organization for confocal microscopy. Both protocols have consistently worked very well for me for a variety of cells and fluorescent probes.

Glutaraldehyde 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 the cells with BRB80 buffer (80 mM PIPES, pH 6.9; 1 mM MgCl2; 1 mM EGTA) to remove medium and serum proteins.
3. Fix cultures with 0.3% glutaraldehyde in BRB80 for 10 minutes at room temperature.
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 2 more times.
7. Wash the cells with 3 brief rinses with a solution of 0.1% Triton X-100 in PBS (PBST).
8. If cells are to be stained with antibodies, block the coverslips with a solution of 5% normal goat serum in PBST for 20 minutes to reduce non-specific binding.
9. 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..
10. Remove unbound antibodies by washing the cells 3 time for 10 minutes each in PBST.
11. For immunofluorescence, dilute an appropriate fluorescently-labeled secondary antibody according to the manufacturer's stipulation into 5% normal goat serum in PBST. Apply secondary antibody solution to the coverslips as stated in step 9, above. Stain for 1 to 2 hours.
12. Remove unbound secondary antibodies by washing the cells 3 times for 10 minutes each in PBST.
13. If a non-antibody fluorescent stain is to be used, dilute it into PBST and apply to coverslips as outlined in step 9, above. For labeling filamentous actin, dilute fluorescently-conjugated phalloidin according to manufacturer's guidelines and stain for 20 minutes. For labeling DNA, dilute DAPI to 10 ug/mL into PBST (from a 1 mg/mL stock in water) and stain for 20 minutes.
14. Wash 3 times, briefly, with PBS (no detergent) and one time with de-ionized water.
15. 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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