Rinse after Block and Permeabilize

Steps A-D and J-L that were covered in Chapter 10 for the single antibody procedure are the same and will not be repeated here. The discussion below will focus on the steps necessary for antibody incubations from D through I. Because both E antibodies and 2° antibodies can be combined in single incubation steps, the number of steps and the amount of reagents will be similar to those in the single 1° antibody procedure. 

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. 

The two 1° antibodies are made in different species and can be combined in one incubation solution. The mouse anti-Ag A at 1 500 and the rabbit anti-Ag B at 1 1000 are mixed in a single solution. For the 17 samples in this experiment, but not the three 2° control conditions, use 250 p.1 per sample, plan for 4.25 ml plus 10% excess or 4.7 ml of the rinse buffer. The mouse anti-Ag A at 1 500 will be 

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