Abstract
A novel Histo-ELISA technique is intended to facilitate quantification of target tissue proteins in a tissue section and involves the selection of target regions in the tissue section, application of streptavidin-conjugated HRP (horseradish peroxidase), coupled with peroxidase substrate-TMB (3,3',5,5'-tetramethylbenzidine), and staining dye evaluation with ELISA reader. The target protein content (weight per volume unit) was translated from optical densities by a reference standard curve, obtained via parallel staining of the targeted protein-coated slides. To validate the technique, we carried out quantifications of IgG extravasation in ischemic and nonischemic brain sections in a mouse stroke model. With those obtained data and the reference of immunohistochemistry scores assessed on the adjacent sections, accuracy, sensitivity, and precision for the technique were evaluated. For all evaluated parameters, Histo-ELISA performance was either comparable to or better than the standard immunohistochemistry. A comparison with the data from the repeated measurements yielded a rather low coefficient of variation. The results confirmed that the technique is a fairly reliable quantitative test with rather high sensitivity, accuracy, precision, and reproducibility for detecting target protein content in tissue sections and that its tissue distribution and related subsequent morphological changes can be observed at the same time.