Immunofluorescence Yeast

Wednesday, May 05, 2004

Description
Immunofluorescence Yeast

Procedure
1) Grow cells in YEPD or defined media to a concentration of 1-5 X107(protocol is for ~5 ml of exponentially growing cells).
2) Add 0.6 ml of 37% formaldehyde solution directly to the cells andincubate with gentle shaking for 90 min at the same temperature as growth.
3) Transfer cells to a 15 ml conical tube and pellet by centrifugation(3 min @ 2000).
4) Aspirate supernatant and wash cells with 5 ml 0.1 M KPO4, pH 6.5.
5) Repellet cells and wash with 5 ml 0.1 M KPO4, pH 6.5/1.2 M sorbitol(P solution).
6) Pellet cells and aspirate supernatant. Resuspend in ~1 ml P solution.
7) Add 25 l of DTT for 10 min then 25 l of zymolyase. Incubate cells on the roller at 30C for one hour (or until digestion is sufficient).
8) Pellet cells and aspirate supernatant. Resuspend in ~1 ml of P solution.
9) Place 20 l of cell suspension into each well of a prepared slide.After a few minutes aspirate excess cells.
10) Immediately immerse slide in ice-cold methanol for 6-7 min. Remove and immerse in ice-cold acetone for 30 s. Allow slides to air dry.
*Once cells are fixed- try and keep the wells moist at all times.
11) Wash slides once with 10 mg/ml BSA in 1X PBS. Remove supernatant and add 1 antibody (diluted in BSA/PBS). Incubate in a moist chamber for at least 2 h.
12) Aspirate excess solution and wash 4X with BSA/PBS. Add 20 l of 2 antibody (diluted in BSA/PBS). Place in dark/moist chamber for ~2 h.
*Remember to keep slides covered to prevent bleaching of the 2 Ab.
13) Aspirate excess and wash 3X in BSA/PBS. Then wash once with 1X PBS. Dilute DAPI (1:1000) in 1X PBS. Add DAPI to wells and allow to incubate a few minutes. Aspirate excess, wash once with 1X PBS, and allow slides to air dry.
14) Cover wells with anti-fade reagent and cover slip- seal with clear nail polish.

Recipes
0.1 M KPO4, pH 6.5
0.1 M KPO4, pH 6.5/1.2 M sorbitol (P solution)
zymolyase- make a 10 mg/ml solution in P solution, vortex and let it sit on the bench for 5 min. Then spin in fuge for 5 min and use clear supernatant
10 mg/ml BSA in 1X PBS
1X PBS
antifade solution- p-phenylenediamine (1 crystal in 100 l 10X PBS then add 900 l glycerol)

Supplies

Tips
Slide Preparation-

Coat slide wells with 0.1% polylysine. Aspirate after 10-30 s. Wash each
well with water three times. Allow the slide to air dry for 10 min

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Immunofluorescence Yeast