Intracellular Staining

(If you have to do surface staining beware that, certain epitopes loose their reactivity after fixation, in this case start by performing the surface staining, wash and then continue with the subsequent steps, if the Ab does not loose reactivity after fixation add it together with the intracellular Ab ).

Pellet cells

Fix by resuspending in 200µL PBS 3.7% Formaldehyde, 10' RT

wash by adding excess PBS, pellet 5' 1500-1800 rpm (increasing the speed might avoid loosing cells after fixation with centrifugation)

Permeabilize by resuspending in 100µL PBS 0.1 Triton X-100, 5'R

wash by adding excess PBS, pellet 5' 1500-1800 rpm

Block by adding 100µL PBS-2%FCS 10'RT

Add antibody in 100µL PBS-2%FCS (anti-kappa, anti IgM-H, anti IgG-H all 1:500), 15'RT

wash and resuspend in PBS

keep at 4°C

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