TNB blocking buffer: 0.1M Tris HCl, 0.15M NaCl and 0.5% blocking reagent (Roche 11096176001) (Mix 0.5g blocking reagent in 100ml of 0.1M Tris HCl /0.15M NaCl. Heat at 60°C with continuous stirring for 1h. Aliquot and store at -20°C)
Alexa conjugated secondary species specific antibody (Alexa Fluor A21434, A21202, A11059 Thermo Fisher)
Put the 13mm diameter coverslips into a 6 well plate (3-4 coverslips per well) and place under U.V. light for 30 min.
Plate adherent cells directly on the coverslips. For non-adherent cells cover the coverslips with poly-L-Lysine and leave for 1h at 37°C. Remove the poly-L-Lysine and wash once with PBS. Plate 1x106cells/ml per well and leave for 30 min in the incubator or overnight (making allowances in the dilution any cell division that will occur). Do all these steps under sterile conditions.
Without removing the medium add the same amount of 8% PFA (in distilled water) to fix the cells at a final concentration of 4%. Leave for 10 min at room temperature.
If necessary, permeabilize the cells with Triton X-100 (0.5% maximum) in PBS for 5 min at room temperature.
Wash 2 times with PBS and incubate with TNB for 30-60 min at 37°C min to avoid any non-specific binding by antibodies.
Remove the TNB without washing and add 150μl of a primary antibody (1-10μg/ml) diluted in TNB (or neat supernatant). Incubate at 37°C for 30 min.
Wash the coverslips 3 times with PBS. Add the Alexa-conjugated secondary antibody (1:200) and the DAPI (1:100) at the same time, both diluted in PBS and incubate for 30 min at room temperature.
Wash 3 times with PBS and place 10μl of Prolong mounting medium on the coverslip. Place the coverslip with the cells face down on a glass slide.