![]() Histo-ELISA combines the ease, speed, and morphological information gained from classical IHC and a robust quantitative assessment of proteins in situ borne in conventional ELISA.įor the validation of the method, parameters such as sensitivity, limit, accuracy, precision, and repeatability have been assessed using quantification of Immunoglobulin (IgG) infiltration in a stroke mouse model. 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. In this article, 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. Accurate and robust measurement of target proteins in the intact tissues, without loss of morphological information, is a challenge. ELISA techniques or Western blotting often requires sample pooling to obtain a specimen sufficient for accurate analysis and lacks the profiles of relative tissue morphological change and target protein microscopic distribution. Quantification of intra- or extra-cellar proteins in small pathological specimens is difficult with the other conventional methods. In addition, changes in chemicals, working temperature, section thickness, and reagent reaction duration can lead to a significant Lab-to-Lab or observer-to-observer variations on a classic IHC stain, which is reflected in the form of lower or higher staining intensity 6. However, traditional IHC is semi-quantitative at best in the sense of scientific strictness 1, 2, 3, 4, 5. Immunohistochemistry (IHC) based methods are widely used to detect target proteins in different studies due to facts of ease of use, turnaround time, cost, and morphological information. 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. A comparison with the data from the repeated measurements yielded a rather low coefficient of variation. For all evaluated parameters, Histo-ELISA performance was either comparable to or better than the standard immunohistochemistry. With those obtained data and the reference of immunohistochemistry scores assessed on the adjacent sections, accuracy, sensitivity, and precision for the technique were evaluated. To validate the technique, we carried out quantifications of IgG extravasation in ischemic and nonischemic brain sections in a mouse stroke model. 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. 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.
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