Rinse After 2 Antibody

After incubation with combined 1° antibodies, all of the antibody must be rinsed away. Each rinse will be in 500 p.1 of rinse buffer and a total of four rinses for 5 min with agitation. 

During all rinses, as stated previously, the key is to leave solution on the sample, which will prevent drying of the sample and thus prevent drying of the section. After each rinse, leave 10-20% of the liquid on the sample and do not remove all of the solution. Drying of the sample will dislodge cells on the coverslip and permanently attach antibodies to the cells which increases background. A minimum of six 5-10 min rinses are recommended. Many labs use as much as 10 rinses to remove all of the antibody from the previous step 

As with the D antibodies, the 2° antibodies can be combined into one incubation solution, with goat anti-rabbit 555 fluorophore at 1 1000 and the goat anti-mouse 488 fluorophore at 1 1000. Both of these antibodies are diluted in a single buffer solution. For the 20 samples in this experiment at 250 xl per sample, plan for 5 ml plus 10% excess or 5.5 ml of the rinse buffer. The goat anti-mouse 488 fluorophore at 1 1000 will be 5.5 xl of stock and the goat anti-rabbit 555 fluorophore at 1 1000 will be 5.5 xl. The incubation with the D antibody is done for 1 h on a rocker at room temperature. 

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G) Rinse after 1° antibody - After 1° antibody incubation, all of the antibody must be diluted and washed away. Each rinse will be in 500 xl of rinse buffer and a total of six rinses, each done for 10 min with agitation. To correctly perform rinses, the key is not to totally remove all solution from the sample, which can cause drying of the sample. Rather, keep the number of rinses high. With a sufficient number of planned rinses, leaving 10-20% of the liquid at each rinse will allow removal of all of the previous incubation solution. Many labs are using as much as ten rinses to remove all of the antibody from the previous step... 

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. 

Fig. 10.3 Rinses after the 1 ° antibody before the 2° antibody. Images show results after normal processing and incubation with the 1° antibody and varying numbers of rinses before a normal incubation with the 2°. (a) With one rinse, the specific labeling was low and no background was seen, (b) With three rinses, the specific labeling was still low and no background was seen, (c) By five rinses, the level of specific labeling increased, (d) With seven rinses, the level of specific labeling was high and there was no background. Mouse spinal cord was processed as in Fig. 10.2. The wells for the incubations were 80-100 p.1 in volume...

I) Rinse after 2° antibody - After 2° antibody incubations, a total of four rinses will be done with two different buffer solutions. First, rinse twice with 500 p,l of rinse buffer for 5 min with agitation. Next, rinse twice with 500 p,l of PBS alone for 5 min with agitation. The last buffer rinses are needed alone because some mounting media interact with proteins in the rinse buffer forming precipitates. 

A new Experimental Design Chart must be used for this Indirect Immunocytochemistry Block-Between (Table 12.1), because it differs from the chart used previously. Here, the experiment uses cultured cells on coverslips and two 1° antibodies made in mouse, mouse anti-Ag A and mouse anti-Ag B. A new row on the Chart is needed, 4. Block-between, to list two new reagents used to block-between the two 1° antibodies. Normal mouse serum is used at 1 20, which is sufficient to block in most cases. The anti-mouse Fab molecule is used at 20 p.g/ml. Incubations for each of these steps is for 1 h with six rinses after each incubation. Note these blocking incubations must be performed in the order of normal mouse serum first and anti-mouse Fab second. 

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