Immunofluorescence in yeast-酵母免疫荧光
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Spin 2 ODU of cells 3000rpm, 5'
Resuspend in 5ml of 0.1M KPi pH 7.0
Add 0.6ml 37% formaldehyde and rotate (on nutator) at r.t. for 2 hrs.
(can store cells fixed at 4'C O/N)
Spin down cells (2500 rpm, 2')
Wash 2x in 5ml 0.1M KPi pH 7.0
Wash 1x in 5ml 1.2M Sorbitol in 0.1M KPi pH 7.0
Resuspend in 1ml 1.2M Sorbitol/0.1M KPi pH 7
Add 5ul B-mercaptoethanol and 45ul of 10 mg/ml zymolyase
Incubate 45' in 30'C incubator (shake tubes once in a while)
Spin and wash cells in 5ml 1.2M Sorbitol/0.1M KPi pH 7
Resuspend cells in 1ml 1.2M Sorbitol/0.1M KPi pH 7
(can store cells like this at 4'C O/N )
Staining:
Clean slides in ddH2O and acetone.
Dilute 1% (w/v) polylysine 10-fold in water
Put 15 ul dil. polylysine per well for 5' (Pour MeOH and acetone into
Coplin jars at -20'C)
Aspirate off liquid w/ drawn out Pasteur
Wash 3x in distilled water
Add 15 ul of fixed cells to each well for 10' (r.t)
Aspir. liq. and plunge immed. into -20 MeOH for 6' then acetone (-20)
for 30 seconds.
Air dry slides.
Put 1 drop BSA/PBS (1x PBS + 10 mg/ml BSA) in ea. well for 5' (r.t)
Aspir. liq.
Dilute primary Ab in BSA/PBS
Add 15 ul of primary Ab to each well
Incubate slides in moist chamber for approx. 2 hrs (r.t)
Wash wells (by aspiration) 4x with BSA/PBS
Dilute secondary Ab in BSA/PBS
Add 15 ul of secondary Ab to each well
Incubate slides in moist chamber in dark for 2 hrs. (r.t)
Wash 4x in BSA/PBS
Air dry slides
Add mounting medium to slide
1 mg phenylenediamine(-prevents quenching of fluor)
first add 100 ul 1M KPi ( monobasic) then after it goes into solution, add
900 ul of glycerol (100%)
Coverslip the slide and seal with nail polish. Store at -20 C
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Resuspend in 5ml of 0.1M KPi pH 7.0
Add 0.6ml 37% formaldehyde and rotate (on nutator) at r.t. for 2 hrs.
(can store cells fixed at 4'C O/N)
Spin down cells (2500 rpm, 2')
Wash 2x in 5ml 0.1M KPi pH 7.0
Wash 1x in 5ml 1.2M Sorbitol in 0.1M KPi pH 7.0
Resuspend in 1ml 1.2M Sorbitol/0.1M KPi pH 7
Add 5ul B-mercaptoethanol and 45ul of 10 mg/ml zymolyase
Incubate 45' in 30'C incubator (shake tubes once in a while)
Spin and wash cells in 5ml 1.2M Sorbitol/0.1M KPi pH 7
Resuspend cells in 1ml 1.2M Sorbitol/0.1M KPi pH 7
(can store cells like this at 4'C O/N )
Staining:
Clean slides in ddH2O and acetone.
Dilute 1% (w/v) polylysine 10-fold in water
Put 15 ul dil. polylysine per well for 5' (Pour MeOH and acetone into
Coplin jars at -20'C)
Aspirate off liquid w/ drawn out Pasteur
Wash 3x in distilled water
Add 15 ul of fixed cells to each well for 10' (r.t)
Aspir. liq. and plunge immed. into -20 MeOH for 6' then acetone (-20)
for 30 seconds.
Air dry slides.
Put 1 drop BSA/PBS (1x PBS + 10 mg/ml BSA) in ea. well for 5' (r.t)
Aspir. liq.
Dilute primary Ab in BSA/PBS
Add 15 ul of primary Ab to each well
Incubate slides in moist chamber for approx. 2 hrs (r.t)
Wash wells (by aspiration) 4x with BSA/PBS
Dilute secondary Ab in BSA/PBS
Add 15 ul of secondary Ab to each well
Incubate slides in moist chamber in dark for 2 hrs. (r.t)
Wash 4x in BSA/PBS
Air dry slides
Add mounting medium to slide
1 mg phenylenediamine(-prevents quenching of fluor)
first add 100 ul 1M KPi ( monobasic) then after it goes into solution, add
900 ul of glycerol (100%)
Coverslip the slide and seal with nail polish. Store at -20 C
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