Comparison of contrast, sensitivity and efficiency of signal amplified and nonamplified immunohistochemical reactions suitable for videomicroscopy-based quantification and neuroimaging

Abstract

In recent years, many different technical modifications of immunohistochemical methods have been developed. The selection of a suitable technique for quantitative purposes such as mapping studies can be quite difficult. Various features of a certain method must be considered such as the sensitivity, costs, duration and practicability with respect to serial sectioned specimens. Background and foreground difference or contrast and the influence of artifacts are major problems of quantitative immunohistochemistry. It is not known which of the different modifications of immunohistochemical signal amplifications and non-amplifications gives optimal results in respect to image analytical-based quantification. However, for image analysis, it is important to analyze sections which offer a sufficient contrast between foreground and background. The sensitivity of a system is crucial when quantitative immunohistochemistry should be applied to scarce material with longer postmortem and storage times which occur often by processing human brains. In addition, the enzyme–substrate reactions have an obvious influence on this criterion; therefore, different substrates were also tested. The contrast may be as well effected by the quality and specificity of the primary antibody, the type of tissue and naturally by preparative (fixation, postmortem delay, storage) and individual factors (age, circadian effects, diseases, sex). Because all of these factors may yield to different results by combining them with different neuronal structures, we used three different antigen expressions for a specific analysis: fibrillary, granulary and perikaryal antigen distributions in brains from Wistar rats.

Principally, the sensitivity of the modifications of immunohistochemical amplifications is revealed more strongly than without enhancement steps; however, the contrast between foreground and background structures does not necessary increase by applying a certain amplification technique. The lowest contrast (15%) was detected after applying the labelled streptavidin–biotin technique. All other methods offer comparable contrasts in between 30% and 40%. The catalyzed signal amplification reaction has been found to give optimal results (40% contrast) for image analysis. However, from the technical point of view and variability of protein expression, storage and postmortem delay, it was necessary to adapt the commercial CSA Kit from Dako (K1500). The modified technique, called C2 method, offers better results with respect to sensitivity, total costs, duration and contrast (60%) and variability of contrast.