Nonspecific staining

Hebert, G.A., Pittman, B., and Cherry, W.B. (1967) Factors affecting the degree of nonspecific staining given by fluorescent isothiocyanate labeled globulins./. Immunol. 98, 1204—1212. 

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 ... 

In addition to the many enzyme systems available, there are with each a series of chromogenic substrate solutions that can be used to create different colors and locations of reaction products. For the peroxidase system, there are numerous oxidizable compounds that precipitate as a permanent color. The most common and still widely used is 3,3 diaminobenzidine tetrahydro-chloride (DAB). This compound precipitates to a golden brown color when in solution with peroxidase and hydrogen peroxide. This brown color has many subtleties and readily stands out in a tissue section. With practice, it is possible to differentiate specific from nonspecific staining patterns just by examining the characteristics of the precipitated pigment. This material is also insoluble in alcohol and xylene, and therefore the tissue may be routinely dehydrated and cleared without loss of chromogen. 

Among the sandwich techniques, the ABC is the most universal, with the PAP being the least prone to nonspecific 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 for occasional problem specimens, will most often suffice. 

If the nonspecific staining is high, 0.1 M EDTA can also be added to the rinse solutions. 

The concentration of the primary antibody can range from 1-20 pg/mL with 5 pg/mL being average. If nonspecific staining is a problem, the antibody may be dilated in high-salt Tween-20 bnffer. 

Improper fixation Poorly fixed specimens exhibit intense nonspecific staining. If specimen is too thick to permit complete fixative penetration, inadequate preservation will result. 

It has been reported that incubation of cells in 0.1% sodium borohydride in 1 mL PBS for 30 min at room temperature, after the antibody incubations, reduces autofluorescence and nonspecific staining This has not proved to be advantageous in the author s hands... 

The amplification power of TSA-ABC can be enhanced by using several subsequent cycles of incubation or by extending the duration of incubation, for example, up to 30 min at 37°C. Longer incubations may result in background noise (nonspecific staining). Also, the number of cycles possible before the background noise level becomes unacceptable is... 

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. 

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... 

The presence of this artifact poses a distinct risk of its being interpreted as positive staining, as the artifact can be intense and may be precisely located in the cells of interest with a clean background. It is known that liver and kidney can retain high amounts of retrievable biotin-avidin activity in neoplasms. The need for adequate controls or biotinblocking procedures is obvious when histochemical or immunohistochemical procedures are used. Negative controls facilitate identification of such nonspecific staining. 

Extracts from —3 X 107 cells and a mixture of GM, and GMg were compared by TLC after staining with resorcinol. To improve sensitivity of technique, the spray was applied heavily, giving rise to some nonspecific staining. Only those bands that reproducibly stained the characteristic blue of gangliosides are bracketed. 

The third problem that needs to be controlled is that of spectral cross-over and the possibility of incorrect instrument compensation. As an example of a case in which controls for nonspecific staining, autofluorescence, and compensation are all critical, let us look at the staining of B lymphocytes for the CD5 marker present with only low density on their surface. As well as the problems created by nonspecific staining and by autofluorescence, the problem of spectral cross-over between fluorescein and phycoerythrin can particularly confuse the interpretation of results from this kind of experiment. Look at Figure 6.5. What we are interested in is the number of B lymphocytes that possess the CD5 surface antigen. These cells will appear in quadrant 2 of a contour plot of fluorescein fluorescence... 

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). 

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