Background labeling

Fig. 6.1. Equilibrium labeling for optimal discrimination of mutant clones. At high ligand concentrations, both mutant and wild-type cells are labeled to saturation at low ligand concentrations, both exhibit background labeling. At an intermediate ligand concentration, the ratio of mutant to wild-type fluorescence is maximal.

All antibody solutions should be clear and free of particles or other precipitated material essential to eliminating background labeling in immunocytochemistry. IgG purification removes any particulate material from the whole serum, supernatant, or ascites fluid that could cause background. 

Initially, look at an experimental to determine whether the 2° antibody labeling is as expected. Next, examine the 2° antibody control section to confirm no labeling. Finally, determine that there are no other labeling problems (e.g., background labeling) and that the level of labeling is acceptable. If there are problems with these conditions, then repeat the experiment with corrective measures (Chapter 14, Troubleshooting). Otherwise proceed to evaluate the results of the experiment and determine if the results meet your scientific expectations. 

Hypothesis generation will include things that could give low levels of specific labeling or high levels of background label. The following is a potential list of problems ... 

Background labeling is random labeling at lower level than specific labeling and it can take a variety of forms. Most of the times background is diffused and fairly uniform, but sometimes it is particulate and irregular. A list of potential causes includes... 

A major limitation of the postembedding electron microscopic immunocytochemistry is the lack of penetration of the antibodies and label into the section. For densely concentrated antigens, this is not a limitation. For example insulin in secretory vesicles will have sufficient insulin antigen exposed on the surface of the section to label the vesicle (Fig. 15.4b arrows). However, infrequent cytoplasmic proteins may not have enough antigens exposed on the surface that will be seen over background labeling. 

Longer incubations permit the use of lower dilutions of pnmary antibody, resulting usually in reduced nonspecific background labeling. The optimal dilution of primary antibody must be ascertained as detailed in Note 27. 

The cleanest signals are generally obtained using antibodies labeled with fluorescine isothiocyanate (FITC) or carbocyanine dyes (Cy 3). If possible, avoid antibodies labeled with rhodamine (TRITC) or, to a lesser extent, Texas red, since in our hands at least, these tend to yield higher levels of background labelling For Vibratome sections, increase the incubation tune to 3-4 h. For retina or other wholemounts, incubation times on the order of 8 h may be necessary. Likewise, for cultured cells, secondary antisera incubation tunes may need to be extended, this will have to be determined empirically. 

It IS imperative that the slides are not allowed to dry out completely from this step onwards, otherwise there will be unspecific background labeling. 

A frequently encountered problem is to distinguish between low and diffuse (ubiquitous) signal and background labeling In such cases, competition experiments using at least 10- to 20-fold excess of unlabeled antisense probe may be performed, which should compete only for specific binding. 

Three different label patterns exist with zero, one or two different non-background labels in the neighborhood of an object pixel which are called new label pattern, label copy pattern, and merger pattern. They induce a new label operation, a label copy operation, or a merger operation. 

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