Koshland Web Site/Methods



Iindirect immunoflourescence for yeast:
  1. 1mL yeast (~0.2-0.5 OD) + 0.1mL 36% formaldehyde, 60'/Rt
  2. Wash 2x 1mL H2O (spin 10K, 5"). Wash in 1mL SK and resuspend in 450 ul SK. Sonicate 5 sec. lowest setting.
  3. Add 50 ul fresh 10x digestion sol -- 10-15'/Rt *microscope: cells not dark but halo dimmed
  4. Cells a little under done, add 10 ul/polylysine well. -- 5'/Rt. Aspirate. (Wash with H2O (10 ul)) skip
  5. Drop slides -20oC MeOH -->6'. then -20oC acetone/30". Dry. (can store 4oC)
  6. Add 15 ul 10% BSA in PBS/well. 20'/Rt (humid box). Aspirate.
  7. Add 15 ul 1/200 YOL1/34 (rat a tubulin) in 10% BSA/PBS --60'/Rt
  8. Aspirate (do not let dry) and wash 5x 15 ul PBS
  9. Add 15 ul 1/200 goat alpha rat-FITC --60'/Rt (dark, humid box)
  10. Aspirate 2d and wash 5x 15 ul PBS
  11. 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
    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 (???/???)**

    **VG uses oxaliticase, works better!!! for some IIF???
Comments: Standard method good for tubulin and spindles
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