Wittrup: Fluorescence Microscopy of Yeast
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.
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.
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