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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.
Highlights
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, coupled with peroxidase substrate—TMB (3,3′,5,5′-tetramethylbenzidine), and staining dye evaluation with ELISA reader
Level of IgG extravasation into the brain and related morphological changes are important indices to judge the degree of blood–brain barrier damage during cerebral ischemia[7,8,9] and there are no methods currently available to do both in one intact tissue block at the same time
Laboratory daily routine works on the path-histology resulted in the development of Histo-ELISA—a method for Quantitative Evaluation of Tissue Components in Locality
Summary
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. 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. We describe a Histo-ELISA technique, a fusion method of conventional ELISA, and standard ABC immunostaining to quantify and localize target proteins and to observe the related morphological changes concurrently. It is based on the application of peroxidase substrate—TMB (3,3′,5,5′-tetramethylbenzidine) instead of DAB (3,3′-diaminobenzidine) and ELISA reader evaluation at the end of classical immune staining. With the Histo-ELISA, we quantified IgG infiltration and observed pathological changes spontaneously and by the way, the effectiveness of revacept on the stroke was evaluated in the ischemia models
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