Methods & Techniques
For Histologists and Immunohistochemists		  

Cell Biology Applications of Fluorescence Microscopy

 

Stephen Rogers

Manager, Microscopy Suite, Light and Confocal Microscopy

Imaging Technology Group
Beckman Institute for Advanced Science and Technology
University of Illinois at Urbana-Champaign
405 N. Mathews, Urbana, IL 61801

Sample Fixation Protocols

 

Below, I have provided example protocols for immunofluorescence staining of cultured cells. These protocols were adapted from Bacallao and Steltzer 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.

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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