Block and Permeabilize

For this example experiment, cell cultures were prepared on 12 mm glass coverslips in a 24-well plastic culture dish. 

The culture medium is removed and the cultures are rinsed twice in PBS. The PBS is removed and the fixative, 4% paraformaldehyde in PBS, is added for 30 min. 

Blocking and permeabilization solution contains 10 mg/ml BSA, 5.0 % normal goat semm to block nonspecific antibody binding and 0.02% sodium azide is used to prevent bacterial growth during incubations all in PBS. Perform permeabilization with 0.2% Triton. Incubate for 1 h. 

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The rinse buffer solution contains 10 mg/ml BSA and 5.0% normal goat serum in PBS (no detergent) use this for all rinses. Mix only as much as needed for the current experiment. To prepare this solution, multiply the number of samples times the volume of each rinse, times the total number of rinses for the entire experiment. For this example experiment, we have 17 experimental samples and 3 controls samples for 20 total samples. For the rinse buffer, use 500 p.1 for each rinse and a total of 14 rinses (two rinses after block and permeabilize six rinses after 1° antibody six rinses after 2° antibody). A total of 20 samples x 14 total rinses x 0.5 ml per rinse = 140 ml of rinse buffer plus 5% excess (7 ml) is 147 ml. The excess amount is a safety factor, in case of a spill or other unexpected event. 

The rinses after the block and permeabilize step is to remove the Triton so that it will only act for a specific period of time. Two rinses are sufficient to dilute the Triton. Rinse for 5 min with the same agitation used during the block and permeabilize step. 

Fix cells with 2% paraformaldehyde followed by blocking and permeabilization. 

Cells are next blocked and permeabilized by incubating for 30 min with PBS containing 10% fetal bovine serum and 0.05% saponin (blocking buffer). We find manipulation of the coverslips is easiest by performing all labeling steps on parafilm on the bench top. Covershps are then inverted onto 50-100 yX drops of blocking buffer into which primary antibody has been diluted and incubated for 30 min. 

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