Wittrup: Fluorescence Microscopy of Yeast

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Indirect Immunofluorescence Microscopy of Yeast Cells

Cell Fixing and Permeabilization

1. Induce protein expression through growth (usually overnight) in 5 mL SG-CAA or YPG.

2. Add 500 μl of formaldehyde directly to the culture and shake additional 1.5 hours at 30°C.

3. Remove 1 OD600 of cells and wash twice in 1 mL PBS/0.5% Tween-20 (PBST).

4. Resuspend cells in 0.5 mL 50 μg/mL zymolase diluted in PBST. (2 μl stock zymolase for every 0.5mL of PBST). Incubate 20 minutes at 37°C. Do not overincubate as proteolytic contaminants in the zymolase can ruin your sample.

5. Wash three times in 1 mL PBST. From this point, spin cells no faster than 6000 rpm to avoid breaking open the spheroplasts.

Cell Staining

1. Resuspend the cells in dilution of primary antibody(ies) in 20 μl of PBS/4%BSA. (Use 10 μg/mL chicken anti-myc, 10 μg/mL M2 anti-flag, and 20 μg/mL 13D11 anti-vacuole membrane).

2. Incubate one hour at room temperature.

3. Wash one time in 500 μl PBS/BSA then incubate fifteen minutes in 500 μl PBS/BSA.

4. Wash one more time in PBS/BSA then resuspend in secondary antibody diluted in 20 μl PBS/BSA. (40 μg/mL of Alexa488-conjugated anti-chicken antibody, 10 μg/mL of goat-anti-mouse PE and 1mg/mL Hoechst DNA stain).

5. Incubate one hour at room temperature in the dark. Wash and incubate in PBS/BSA for fifteen minutes as described above.

6. Spin down, resuspend in ~5 μl of PBS.

Slide Preparation

1. Wash slides and cover slips in watch glass with 1 M HCl for five minutes. Wash with water and then wash with EtOH. Dry in fume hood on ChemWipes.

2. Add 20 μl poly-L-lysine to slide and let sit for 1 minute before aspirating off.

3. Wash poly-L-lysine spot with 3 washes of 20 μl PBS. Aspirate off each time.

4. Add 5 μl of cells to lysine spot. Let sit for 1 minute and then aspirate off liquid.

5. Wash cells one time with 15 μl PBS.

6. Add 3 μl of mounting solution and attach cover slip. Apply nail polish to cement slip to slide.

Andy Rakestraw May 5, 2006