Immunostaining antibody concentrations

FIGURE 4.1. Effect of antibody (PC 10) concentration on the immunostaining of PCNA antigen in rectal mucosal proliferating cells. (A) The antibody was used at a concentration of 1 100. (B) The antibody was used at a concentration of 1 400. Note the increased labeling, especially in the upper crypt, when antibody concentration of 1 100 was used. Arrows indicate the upper limit of labeled cells. Reproduced, with permission, from Holt et al. (1997). Copyright 1997 American Association for Cancer Research. 

The pattern of immunostaining will depend on the presence of neoplastic cells, the location (e.g., cell membrane, cytoplasm, or nucleus), proper fixation, background staining, and antibody concentration. Immunostaining for any antigen is rarely uniform in nature. Heterogeneity of immunostaining is the rule rather than the exception, and it is proper to state the pattern, cell localization, and distribution of positive... 

Further dilute (if required) the resultant primary antibody Fab fragment complexes to optimal working concentration (usually about 1 5 pg/ml) in staining buffer containing 10% normal serum and then apply to the sample for 30 60 min at room temperature and proceed further with your standard immunostaining protocol. 

Control slides of known reactivity should be run with each set of slides immunostained. Appropriate controls should include an irrelevant antibody of the same immunoglobulin class at an equivalent concentration. 

FIGURE 6.14. Effect of pretreatment with 1% sodium dodecyl sulfate (SDS) on immunostaining of cultured epithelial cells with anti-AE1/2 affinity-purified antibody at a final concentration of 0.45 i,g/ml. (A) In the absence of SDS treatment, AE2 is barely detectable in MDCK cell cultures. (B) After SDS treatment a bright basolateral membrane staining is visible. Reproduced, with permission, from Brown et al. (1996). Copyright 1996 Springer-Verlag. 

Another method takes advantage of significant differences in detection sensitivities of two immunostaining procedures. For example, the streptavidin-biotin method with biotinylated-tyr-amide amplification can detect antigens at such a low concentration of the primary antibody, that they can t be detected with the traditional indirect methods used in the subsequent immunoreac-tions (38, 39). The principles of double/multiple immunostaining techniques are summarized in Table 3. 

Fig. 2.6 Identification of recombinant clones by direct immunostaining. Colonies were incubated with Texas Red-labeled antibodies against IgC at concentrations allowing the formation of immune precipitates at colonies expressing leptin-Fc...

After autoclaving in TB containing 200 mM NaCl for 10 min, the sections were further treated with TB for 15 min at 95-98°C. All antibodies partially recovered their immunostaining after the second heating (Table 17.3). These results demonstrate that the ionic strength of the solution is one of the critical factors for HIAR, and that a high concentration of salt inhibits the exposure of epitopes, preventing their reaction with antibodies. 

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