PDS1::HA IMMUNOFLUORESCENCE

Monday, May 10, 2004

Description
PDS1::HA IMMUNOFLUORESCENCE

Procedure
Cultures: 1500, 1501
Fixations: 10, 20, 30 and 45 minutes in 4% formaldehyde, 0.1 M KPO4 pH 6.5; 15 minutes in 4% formaldehyde, 50 mM KPO4 pH 6.5, 0.5 mM MgCl2

Grow cells to OD 0.5
Harvest 24 and 6 mls of each culture separately. Resuspend the '24 mls' in 10 ml 4% formaldehyde, 0.1 M KPO4 pH 6.5, and the '6 mls' in 2.5 ml 4% formaldehyde, 50 mM KPO4 pH 6.5, 0.5 mM MgCl2. Put on rotating wheel at 23癈.
At indicated times (-2 minutes for first spin) remove 1.5 mls and wash at room temp X4 with 1 ml 0.1 M KPO4 pH 6.5 (spin 1 min at 10K). (Can keep on ice)
Wash once with 0.1 M KPO4, 1.2 M sorbitol. Resuspend in 1 ml 0.1 M KPO4, 1.2 M sorbitol
Usually I spheroplast 0.5 mL of cells and store rest at 4癈 (just in case). To 0.5 mL of cells, add 0.5 mL of 0.1 M KPO4, 1.2 M sorbitol containing 1 礚 bME and 10 礚 oxalyticase (1 mg/mL stock).
oIncubate at 23癈 wheel; check after 20 min. Cell are spheroplasted when they swell up (explode) when water is added from the side.
While cells are spheroplasting, prepare polylysine coated slides .
Harvest spheroplasts by spinning 3-5 min at 3K. Wash once with 1.5 mL 0.1 M KPO4, 1.2 M sorbitol, using a P1000 to gently resuspend cells--NO VORTEXING. Resuspend washed cells in 0.5 mL 0.1 M KPO4, 1.2 M sorbitol.
Put 20 礚 cells in each polylysine-coated well. Let cells settle 10-20 min. Aspirate sups and immediately (but gently) plunge into -20癈 Methanol for 6 min. Transfer to -20癈 Acetone for 30 seconds.
Allow slides to air dry for 1-2 min. Put 20 礚 PBS-BSA (1X PBS, 1% BSA)on each well. Put slide in humid chamber for at least 5 min.
Aspirate PBS-BSA right before adding primary antibody.
a) Dilute primary antibody in PBS-BSA. Try 1:1000, 1:2000, and 1:3000 for 12CA5.
b) Use 15 礚 aliquots per well.
Incubate at 15癈
Wash wells 3X with PBS-BSA. Apply secondary antibody, (Goat anti Mouse Cy3) 1:1000 . Incubate at R.T. (in the dark) for 2 hr.
Wash wells 3X with PBS-BSA.
Wash 2X with plain PBS.
Aspirate last wash.
Put a drop a mounting medium containing DAPI on each well. Put on cover slip, avoiding bubbles, and seal with nail polish. Store slides at -20癈.

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PDS1::HA IMMUNOFLUORESCENCE