Iindirect immunoflourescence for yeast:

Monday, May 10, 2004

Description
Iindirect immunoflourescence for yeast:

Procedure
1mL yeast (~0.2-0.5 OD) + 0.1mL 36% formaldehyde, 60'/Rt

Wash 2x 1mL H2O (spin 10K, 5"). Wash in 1mL SK and resuspend in 450 ul SK. Sonicate 5 sec. lowest setting.

Add 50 ul fresh 10x digestion sol -- 10-15'/Rt *microscope: cells not dark but halo dimmed

Cells a little under done, add 10 ul/polylysine well. -- 5'/Rt. Aspirate. (Wash with H2O (10 ul)) skip

Drop slides -20oC MeOH -->6'. then -20oC acetone/30". Dry. (can store 4oC)

Add 15 ul 10% BSA in PBS/well. 20'/Rt (humid box). Aspirate.

Add 15 ul 1/200 YOL1/34 (rat a tubulin) in 10% BSA/PBS --60'/Rt

Aspirate (do not let dry) and wash 5x 15 ul PBS

Add 15 ul 1/200 goat alpha rat-FITC --60'/Rt (dark, humid box)

Aspirate 2d and wash 5x 15 ul PBS

Add 5 ul DAPI and antifade mounting medium (power med Rt)

For alphaHA Ab: use 10'-15' Fix critical 1/2000 12CA5 with 1/100 G alpha M C13 for 2d
SK:
7.5 mL 2M sorbitol (25.)
651 ul 1M K2HPO4 (2.17) }50mM
100.5 ul 1M KH2PO4 (335ml)}pH7.5
6.75 uL H2O (22.5)
15mL 50mL

10x digestion buffer:
488ul H2O
0.5 ul B ME
8.5 ul zymolyase

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Iindirect immunoflourescence for yeast: