Rhodamine-Phalloidin/Calcofluor Staining

David Amberg

1. Grow 50 mls of yeast to 5x10E6.
2. Add formadehyde to the media to 4% (33 mls. of 10%). Fix in media at temperature for 10 min.
3. Spin down cells 2-3 K for 5 min.
4. Fix cells in PBS containing 4% formadehyde for 1 hr at temperature.
5. Wash cells 2 times with PBS and reconstitute in 500ul PBS.
6. Remove 100ul cells and add 10ul Rhodamine Phalloidin (6.6uM in MeOH) and add 10 ul 1 mg/ml Calcufluor. Incubate in the dark for 1 hr.
7. Wash the cells 5 times in 1 ml PBS.
8. Suspend the cells in 1 drop of immunofluorescence mounting solution and store at 4¡C (good for a few days).
9. Visualize by placing 2 ul of cell suspension under a standard size coverslip and waiting for capilary action to draw the liquid out thus flattening the cells.

Reagents

Formaldehyde

Polysciences, 10% EM Grade Cat #04018

10XPBS

80 grams NaCl + 2 grams KCl + 14.4 grams Na2HPO4 + 2.4 grams KH2PO4. pH upon dilution to 1X should be 7.2.

Rhodamine Phalloidin

Molecular Probes Inc., Dissolved in methanol according to manufacturers instructions to ~6.6uM and stored dessicated at -20¡C. Cat#R-415.

Calcofluor

aka fluorescent brightener 28. Sigma Cat#F-6259. Dissolve to 1mg/ml in H2O overnight and store at 4¡C in the dark.

Mount

Dissolve 100 mg p-phenylenediamine in 10 mls PBS, adjust the pH to above 8.0 with 0.5 M Na Carbonate buffer (pH 9.0) and bring the volume to 100 mls with glycerol. Add Dapi to 50 ng/ml. Mix thoroughly and store at -20¡C. It turns brown when it is b ad.