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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 a¡ter repeated washes). Fixation: A) Formaldehyde or Para¡ormaldehyde: 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 4ºC at max speed in Eppendor¡ centri¡uge (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 4ºC at max speed in Eppendor¡ centri¡uge (to get rid o¡ any precipitated antibody)...
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- Summer '09
- Th, Genetic linkage, lle le, ﬁsh skin gelatin