Nonspecific staining, causes

In addition, poly-L-lysine is a popular and simple means of adhering a section to the surface of a sMe, but it can cause nonspecific staining with some chromogens ... 

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. 

PBS is also commonly used as a wash buffer for IHC. PBS s advantages are reduced auto fluorescence in immunofluorescent assays, and it is relatively inexpensive compared to Tris-based buffers. However, in some cases PBS can cause higher levels of nonspecific staining, and it has been observed to reduce the specific binding abilities of certain monoclonal antibodies (Anti-CD30, for example). 

However, if contaminating antibodies do interfere with specificity, affinity absorption of the antiserum is usually performed. Batch-absorbed antisera almost always contain residual levels of contaminating antibodies (mostly of the non-precipitating type) and will cause nonspecific staining of tissue if used at excessively high concentration (8). 

A variety of enzymes can be used for incorporation of the label. Klenow fragment of DNA polymerase I is preferable to the DNA polymerase I holoenzyme because it has the identical polymerase activity, but lacks the exonuclease activity that could cause artifactual labeling (43). Alternatively one can use TdT (33), which adds on long tails of nucleotides to the 3 hydroxyl ends of DNA without the need for a template strand (43). It is extremely important to use the correct concentration of enzyme, as increased amounts will lead to nonspecific staining of morphologically normal nuclei (34,35). Obviously, insufficient enzyme will lead to a reduction in the staining of apoptotic nuclei. 

On the contrary, the method has some drawbacks, viz., the use of aqueous silver nitrate solution either with or without the addition of acetic acid, incubation of the test material or sections at 37°C to 56 C, and exposing the sections of H2S prior to treating them with aqueous solution of silver nitrate, not only Increases the possibility of diffusion of AA from its original Q%tu position in the tissue, but also causes nonspecific staining of a number of other reductants besides AA. 

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. 

Nonspecific antibody cross-reactivity with similar or dissimilar epitopes on different antigens may also be the cause of confusing background staining. This is rare however, and can be avoided by using antibodies from hyper-immunized animals or carefully selected clones. 

The findings made with tissue fractionation techniques conflict with histochemical studies of Koenig, who claims that gangliosides are mainly in lysosomes. These discrepancies may result from a variety of causes (1) the staining method used to identify the glycolip-id may be nonspecific and stain sialic acid-containing proteins (2) brain lysosomes may not be separated from microsomes and (3) possibly most of the glycoprotein is in the microsomes, but only that in the lysosomes is detectable by histochemical techniques. 

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