ImmunofluorescenceMicroscopyoftissueculturecells

Immunofluorescence Microscopy of tissue culture cells

These methods are written for direct staining of filamentous actin with bodipy FL-phallicidin and indirect immunofluorescence staining of microtubules with anti-tubulin antibodies. They easily can be modified for conventional immunofluorescence staining of any tissue cultured cell or direct staining of actin filaments

SOLUTIONS

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• PBS:

  10.6 mM Na2HPO4 1.47 mM KH2PO4

  137 mM NaCl  2.68 mM KCl

100% Ethanol, -20 degrees C (stored in freezer)

Tubulin antibody (a mouse monoclonal anti-tubulin antibody)

NDB-Phallacidin (nitrobenzooxadiazole - phallacidin) - binds and stabilizes filamentous actin (F-actin). Phalloidin is a member of the phallotoxin family of bicyclic peptides isolated from the deadly Amantia phalloides mushroom. It is extremely toxic. Since it is such a potent toxin that binds to and stabilizes actin filaments, one of the antidotes to ingesting phalloidin is eating red meat (striated muscle).

We will use BODIPY FL-phallacidin (Molecular Probes #B-607), a water-soluble phallacidin labeled with a bodipy fluorophore that can be detected with standard filters for fluorescein. Bodipy phallacidin is dissolved in 1.5 ml of methanol and kept at -20 degrees C. ContactMolecular Probes for other actin probes.

Antibleach solution for Fluorescence Microscopy [from Johnson et al, 1981, J. Immunol. Meth 43:49]
Carbonate-bicarbonate Buffer, pH 9.2-9.5
0.5M Na₂CO₃.H₂O 10 ml (6.2 g/100 ml)
0.5M NaH₂CO₃ 90 ml (4.2g/100 ml)

1. Dissolve 10 mg p-phenylenediamine in 1 ml PBS on ice with vortexing

2. Add 9 ml glycerol, for a total of 10 ml

3. Adjust the pH to 9.5 with carbonate-bicarbonate buffer.

4. Store at -20deg. Discard when solution turns brown

After completing immunofluorescence staining, wash the coverslip with PBS, drain the PBS, and then add a drop of the antibleach solution. Invert over glass slide, remove excess solution with a kimwipe and put in a dark place overnight. The solution will harden sufficiently to use the coverslip with oil immersion lenses. If it does not harden, put a little nail polish around the coverslip. 
NOTE: FITC does not fluoresce well at pH <9.0, so make certain that the pH is greater than 9.0.

Gelvatol antibleach solution (Not As Good As The Above) [from Rodriquez,J and Deinhardt, F., Virology (1960) 12:316-317)]
Elvanol (Polyvinyl alcohol, 88% hydrolyzed - Aldrich Chemical # 18,463-2)
Glycerol
n-propyl gallate

1. Dissolve 5 g Elvanol in 20 ml of 0.14 M NaCl in 10 mM KH₂PO₄-Na₂HPO₄ buffer, pH 7.2. Stir 16 hr on a magnetic stirrer.

2. Add 10 ml glycerol and stir 16 hours.

3. Centrifuge 12,000 rpm, 15 min, to remove undissolved particles. The pH should be 6-7.

4. Add 1.5g n-propylgallate with 30 ml Gelvatol to the supernate. Stir overnight.

5. Store in small aliquots at -20 degrees C.

METHOD:

1. Wash cells grown on coverslips three times with room temp or 37-degrees-C PBS. Be certain to remember which side of the coverslip contains the cells. This is critical for all of the following procedures.

2. Fix cells by dipping in -20 degrees C ethanol for 10 min.

3. Rinse three times in PBS

4. Put coverslips, cell side up, on parafilm placed on the bottom of a petri dish. For each of the following steps, add 20-50 microliters of solution to the top of each coverslip. You do not want to use any more antibody or phallacidin than necessary because each of these items are exceedingly expensive (the bodipy fl-phallacidin costs $ 200) or difficult to obtain.

5. Incubate in primary antibody for 1 hr at room temp or 37 degrees C.

6. Wash by dipping coverslip in 3 changes of PBS, 5 min/change.

7. Incubate in secondary antibody for 1 hr at room temperature. Keep in dark or low light. We will use a Zymed Texas Red goat x mouse secondary antibody.
   

• To label actin, add bodipy FL phalloidin with the secondary antibody.

8. Wash with PBS - 3 changes, 5 min/change.

9. Put a drop of gelvatol solution on a slide, invert the coverslip (cells down) and carefully lay the coverslip on the slide. Avoid air bubbles. The gelvatol will harden overnight and samples can be stored and viewed later.