Staining background

The presence of even small amounts of natural antibodies in the serum may also produce nonspecific staining. These antibodies result from prior environmental antigenic stimulation. In fact, such antibodies may increase in the titer during immunization of the 

Nonspecific staining can be caused by Fc receptor glycoproteins present on the cell membrane. This problem is more relevant to frozen sections and smears than to tissues fixed with formaldehyde. The problem can be avoided by using F(ab)2 fragments instead of whole IgG molecules (Boenisch, 2001). Complement-mediated binding may also cause background staining in frozen sections when whole antisera is used however, this problem is not very common. 

Endogenous biotin, distributed in a wide variety of tissues, may also cause background staining with biotin-based immunohistochemical techniques. This biotin is especially abundant in liver, whereas it is poor in the central nervous system and adipose tissue. Endogenous biotin activity is more abundant in the cytoplasm and cryostat sections but is also present in sections of paraffin-embedded tissues. This problem is largely eliminated by using streptavidin-based methods or by sequential treatment of sections (prior to staining) with 0.01-0.1% avidin followed by 0.001-0.01% biotin for 10-20 min each. The biotin problem is discussed in more detail later in this chapter. 

Finally, a few published reports indicate that antigen retrieval at extremely high temperatures may result in nonspecific staining. Baas et al. (1996), for example, have reported false-positive results at a very high antigen retrieval temperature using monoclonal 

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Mundegar RR, Franke E, Schafer R, et al. Reduction of high background staining by heating unfixed mouse skeletal muscle tissue sections allows for detection of thermostable antigens with murine monoclonal antibodies./. Histochem. Cytochem. 2008 56 969-975. 

The best stain is then the optimal for that particular primary antibody if you do not observe any nonspecific background staining in control incubations (see below). 

Endogenous biotin may be a cause for nonspecific background staining (see Sect. 5.4). To eliminate this unwanted staining, apply an avidin/biotin block (for instance Avidin/Biotin blocking kit from VECTASTAIN, Cat. No. SP-2001). Usually, paraffin tissue sections are free from endogenous biotin, and this step may be omitted. 

When staining with any of the fluorescent phalloidins, dilute 10 pL methanolic stock solution into 400 pi PBS. To reduce nonspecific background staining with these conjugates, add 1% bovine serum albumin (BSA) to the staining solution. It may also be useful to preincubate fixed cells with PBS containing 1 % BSA. 

Kim SH, Shin YK, Lee KM, Lee JS, Yun JH, et al. 2003. An improved protocol of biotinylated tyramine-based immunohistochemistry minimizing nonspecific background staining. J Histochem Cytochem 51 129-132. 

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