Antibody, dilution penetration

Overdigestion of tissue is common when proteinase-K or triton is used to improve antigen retrieval penetrance of the primary antibody. The easiest correction is to dilute triton solutions and decrease the time of the proteinase-K incubation. Tissue can also be digested when the hydrogen peroxide solution, used to quench endogenous peroxidase activity, is left on too long or is too concentrated. To correct this, check... 

Fig. 8. The same antigen solution (horse serum albumin) was placed in the upper layer of these two simple diffusion tubes. The antiserum used in the right-hand tube was that of the left-hand tube (i.e., anti-horse serum albumin) that had been partially absorbed by the homologous antigen, so that not only the antibody concentration had been decreased, but the proportion of precipitating to nonprecipitating antibodies had also been greatly decreased hence the difference in the appeiu-ance of the precipitation zone smaller density and fuzzy (instead of sharp) leading edge. The antiserum in the left-hand tube had been diluted in normal rabbit serum in order to ensure a similar penetration h in the two tubes. From J. Oudin, in Methods in Immunology and Immunochemistry (C. A. Williams and M. W. Chase, eds.), Vol. 3, p. 125. Academic Press, New York, 1971.

The r antibody incubation can be as short as 6 h or as long as 24 h at a dilution determined by preliminary experiments with fluorescent 2° antibody. All incubations should be done on a rotator to maximize antibody penetration. At the end of the 1° antibody incubation, perform 4-6 rinses in buffer with blocking reagents for 10 min each. 

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