Jakob Suckale: Silver: Immunofluorescence moved to Silver:Immunofluorescence: accidental extra blank

2008-10-14T18:14:35Z

Silver: Immunofluorescence moved to Silver:Immunofluorescence: accidental extra blank

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Revision as of 18:14, 14 October 2008

ChrisBrown at 20:54, 20 September 2005

2005-09-20T20:54:34Z

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==Materials==
* 37% Formaldehyde
* 1 M DTT
* DAPI stock solution (1 mg/ml, 1000x)
* VectaShield mounting medium
* Clear nail polish
* Slides and coverslips
* 0.1 M potassium phosphate buffer pH 6.5
**Solution A: 5.44 g KH2PO4 into 200 ml ddH2O
**Solution B: 6.96 g K2HPO4 into 200 ml ddH2O
**For pH 6.5, combine 68.5 ml (A) with 31.5 ml (B), bring up to 200 ml with ddH2O, test pH and adjust with KOH
*Solution P
**0.1 M potassium phosphate buffer pH 6.5, and 1.2 M sorbitol
**Add 21.9 g of sorbitol to 85 ml potassium phosphate buffer, mix (heat a little) and then top off to 100 ml for 1.2 M sorbitol, filter to sterilize
*Antibody Blocking Buffer
**1 ml 1 M Tris pH 9.0 (0.1 M Tris pH 9.0)
**300 µl 5M NaCl (0.15 M NaCl)
**500 µl FBS (5% FBS)
**300 µl 10% Triton-X 100 (0.3% Triton X-100)
**7.9 ml ddH2O
*Antibody Wash #1
**1 ml 1M Tris pH 9.0 (0.1 M Tris pH 9.0)
**300 µl 5M NaCl (0.15 M NaCl)
**8.7 ml ddH2O
*Antibody Wash #2
**1 ml Tris pH 9.5 (0.1 M Tris pH 9.5)
**200 µl 5M NaCl (0.1 M NaCl)
**500 µl 1 M MgCl2 (50 mM MgCl2)
**8.3 ml ddH2O

==Protocol==
#Grow 5 ml yeast to early log phase (5 x 106 – 1 x 107 cells/ml)
#Move 5 ml culture from glass tube to 15 ml conical. Add 700 μl 37% formaldehyde, seal the tube with parafilm, and incubate at 30°C for 1 h
#Pellet cells, 2 min at 2,500 rpm, wash 2 x 5 ml 0.1 M phosphate buffer and 1 x 5 ml Solution P
#Resuspend pellet in 1 ml Solution P, cells can be stored at 4 °C for a while
#Take 500 μl of your cells, add DTT to 25 mM (12.5 µl) and incubate at 30°C for 10 min.
#Pellet cells, 2 min at 2,500 rpm and resuspend with 500 μl Solution P
#Add 20 μl zymolyase (10 mg/ml) and 10 μl lyticase (Resuspend 50,000 units lyticase (one bottle) in 1 ml ddH2O) for every 500 µl cells. Incubate at 30°C on rotator, 10-20 min is a good time range.
#While zymolyasing, coat slide wells with 0.1% polylysine. Incubate 10 min at RT. Rinse 2x with ddH2O and air dry.
#Pellet cells, 1 min, 2,500 rpm and resuspend gently in 500 μl of Solution P
#Wash 2 x 500 μl of Solution P
#Apply 20 μl cell suspension to each well, incubate 5 min RT
#Gently aspirate off the excess cells and permeablize them with 20 µl 0.5% Triton-X 100 in Solution P for 5 min.
#Aspirate off Triton and rinse 1 x 500 μl with Solution P
#Block cells in antibody blocking buffer for 30 min to 1 h at RT
#Add primary antibody diluted into antibody blocking buffer and incubate overnight at 4°C
#Wash wells (RT):
#*Ab wash #1 for 10 min
#*Ab wash #1 for 30 min
#*Ab wash #2 for 10 min
#*Ab wash #2 for 30 min
#Add secondary antibody diluted in antibody blocking buffer, 1 h RT
#Repeat washes (from step 16)
#Stain nuclei with DAPI solution for 5 min at RT in the dark (DAPI stock = 1 mg/ml, 1000x, dilute in PBS)
#Wash cells 2 x 5 min in the dark with Antibody wash #2
#Aspirate off Antibody wash #2 and let air dry in the dark for a few minutes
#Apply VectaShield mounting medium (prewarmed to RT), apply coverslip, and seal with clear nail polish
#Publish results in ''Cell''

Silver:Immunofluorescence - Revision history

Jakob Suckale: Silver: Immunofluorescence moved to Silver:Immunofluorescence: accidental extra blank

ChrisBrown at 20:54, 20 September 2005