Non-specific staining

Tasaka, K Kobayashi, M Tanaka, T and Inagaki, C. (1984) Rapid purification of monoclonal antibody in ascites by high performance ion exchange column chromatography for diminishing non-specific staining. Acta Histochem. Cytochem. 17, 283-286. 

The first step in immunochemical detection of proteins after electrotransfer is blocking the support with an inert material to inactivate further non-specific binding of protein. The blocking reagent should cover the membranes at those areas where no blotted protein is bound and should not react with any of the reactants of immunochemical detection cascade as indicated by no non-specific staining, i.e., resulting in blank background of the membrane. 

Figure 4a. Anti-Cytokeratin, Clone 34BE12, Dako Code M0630, on human prostate tissue, rinsed with 150 mM NaCI, 0.05% Tween 20, Tris-Buffered Saline, Dako Code S3006, pH 7.6 at 25 °C. Non-specific staining is evident in the lumen and the connective tissue.

In cases where it is necessary to evaluate non-specific binding potentially caused by sources other than the primary antibody, additional patient tissue sections may be stained with selected reagents. For example, tissues may be stained with just the secondary antibody and/or the enzyme followed by application of the substrate/ chromogen. In cases where the suspected non-specific staining may be the result of endogenous enzyme present within the tissue, this can be confirmed by application of the substrate/enzyme only. 

Troubleshooting flow chart Use this flow chart to determine source(s) of non-specific staining when using an immunohistochemical protocol. 

The high detectability of these techniques may be due to the presence of multiple biotin molecules on the proteins. Non-specific staining can be considerable at high concentrations of ABC which may be related to the fact that biotin is an important coenzyme for transcarbamylation and may be present in some tissues to bind ABC. Pretreatment with avidin and biotin, respectively, prior to the serological detection, could remedy this problem in some cases. 

Note that successful application of any ultrasensitive detection system raises the possibility that it may be necessary to retitrate primary reagents to a higher working dilution in order to avoid non-specific staining. ... 

FIGURE 21.12 (A-D) A strong nuclear and cytoplasmic staining of pi 6 in smears prepared from residual material in the vial from Pap tests. (E) Non-specific staining in metaplastic cells. 

Among the sandwich techniques, the ABC is the most universal, with the PAP the least prone to non-specific staining. More sensitivity can be had with labeled avidin methods or the newer more inventive amplified amplifications. A universally applied method with an alternate technique available, like the PAP or labeled polymer methods for occasional problem specimens, will most often suffice for most laboratory situations. 

Non-Specific Staining Method with Enumeration by Microscopy or Flow Cytometry Fluorescence In Situ Hybridization Specific Labeling with Enumeration by Microscopy or Flow Cytometry... 

Figure 2. Immunolocalization of carbonic anhydrase. Sections of the anterior region of Chlamydomonas. prepared as described previously, show the same pattern of gold particle distribution. Significant staining of the cell wall (cw) even in the region of flagella (fl) attachment is apparent. Some non-specific staining of the starch grains was also found to occur with either pre-immune serum or with the polyclonal antibody directed against carbonic anhydrase.

Analytical methods employing paper electrophoresis (for bibliography see Pezold 1961) have been used in lipoprotein research since the introduction of Sudan Black B as a Upid stain (Swahn 1952). Paper electrophoresis is a deceptively simple technique. The analysis of electrophoretic data is more complex. While a number of important studies have been published, it has sometimes been difficult to interpret data showing arbitrary ratios between partially separated and poorly characterized serum lipoproteins whose concentrations were estimated by non-specific staining. 

Blot dry with fine filter paper to prevent the non-specific staining which sometimes occurs after dilution with water (which will change the pH) in washing. 

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