Genetics3 - B Cell Receptor Signaling Indirect...

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Unformatted text preview: B Cell Receptor Signaling Indirect Immunofuorescence Cells: Plate cells on 12 mm Transwell flters. Or Plate cells on 18x18 mm glass coverslips: Dip coverslip in 100% EtOH; Fame; place in 6-well tissue culture plate Plate cells into the well with the coverslip (not too dense or cells will come o later ater repeated washes). Fixation: A) Formaldehyde or Paraormaldehyde: Do not rinse cells; just suck o media completely; work ast! Add 1.5 ml (to top and bottom compartments o transwells) 4% ormaldehyde fxative (SIGMA HT50-1-2) at RT with or 25 min (very gentle shaking). Wash 2 x quick with PBS. Quench with 75 mM NH 4 Cl (1 M stock) and 20 mM Glycine (1 M stock) in PBS at RT or 10 min with shaking. Wash 3 x with PBS. B) Methanol: Do not rinse cells; just suck o media completely; work ast! Add 1.5 ml (to top and bottom compartments o transwells) 20C-cold MeOH or 10 min at 20C. Wash 3x3 min in PBS to rehydrate cells. Permeabilization & Blocking: Block and permeabilize with 1 ml o PBS containing 0.2% Triton X-100, 3% BSA at 37 o C or 30 min. Optimize: i high background or low signal, instead o BSA try 5% serum (o species o secondary antibody) or 5% fsh skin gelatin Primary Ab incubation: or cells on ilters: Use a scalpel to cut out flter in rectangle shape with the upper right corner cut (to help keep track o orientation) Make primary Ab dilution in Permeabilization/Blocking solution spin 5-10 min at 4C at max speed in Eppendor centriuge (to get rid o any precipitated antibody) Add 30ul Ab dilution (as a drop on paraflm diluted), put flter on the drop so that the cell side o the flter is acing down, then add 20ul on the top. (total: 50ul Ab/flter) or cells on coverslips: With the coverslip on paraflm, add 100ul Ab (diluted in Permeabilization/Blocking solution) Incubate at or 4C overnight (or 1-2 hrs 37 C) in humid chamber. Wash 4x5 min with Washing Solution which is PBS containing 0.05% TX-100 and 0.7% fsh skin gelatin (2.5 ml per well). Secondary Ab incubation (protect rom strong light rom now on) Dilute 2 o Ab with Permeabilization/Blocking solution (Jackson Ab: 100x; Alexa Ab: 200x dilution) spin 5-10 min at 4C at max speed in Eppendor centriuge (to get rid o any precipitated antibody)...
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This note was uploaded on 10/08/2010 for the course MCDB MCDB 1A taught by Professor Senghuilow during the Summer '09 term at UCSB.

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Genetics3 - B Cell Receptor Signaling Indirect...

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