PubMed · 7711846
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G L Bratthauer. 1994. The avidin-biotin complex (ABC) method.. https://doi.org/10.1385/0-89603285-x%3A175
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This work aimed to develop a technique to measure stained areas in images from sample tissue sections, namely when the structure of interest does not fill the entire image field of the microscope. We propose a semiautomated computer-assisted image analysis (SACAIA) method in which brightfield color images of 3,3'-diaminobenzidene tetrahydrochloride (DAB)-stained antigens are converted to their blue component and boundaries are delineated to extract the object of interest. The number of pixels of a defined color (elicited by DAB) is counted and used to measure the stained area relative to the total area of the tissue under study. The percentages of area stained with adenosine A(1) receptor were 40.76+/-2.08 and 42.44+/-2.26% for manual analysis and SACAIA, respectively (P=0.582). A strong linear correlation of A(1) receptor quantification was found (r=0.98, P<0.001, and 95% CI=0.97 to 0.99 for manual method; r=0.99, P<0.001, and 95% CI=0.98 to 0.99 for SACAIA method). The extent to which misclassification affected staining quantification was evaluated by Bland-Altman analysis, indicating that this method can be applied accurately to quantify the immunohistochemical staining area (occupied by a specific antigen) in small sample tissues that do not fill the entire image field of the microscope.
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A new cytochemical method was devised in order to visualize Cu2+ ions in the synaptic area after their intracellular penetration during nerve stimulation of the frog neuromuscular junction (NMJ). The motor nerves were stimulated in presence of Cu2+. After total blockade of the neuromuscular junction, the tissue was treated by ferrocyanide, a precipitating agent of Cu2+, and fixed for optical and electron microscopic observation. The oxidoreductase-like catalytic activity of the copper ferrocyanide precipitate was used to amplify the cytochemical staining by a treatment with diaminobenzidine and H2O2, after permeabilization of cell membranes by Triton X-100. At optical level, an intense staining was observed in the synaptic area. Application of d-tubocurarine (d-TC), a selective inhibitor of nicotinic acetylcholine receptors (nAChRs), markedly reduced the staining. No reaction could be observed in absence of membrane permeabilization. These results suggest that Cu2+ was localized in the cytoplasm of muscle cells after its penetration through nAChRs. At electron microscopic level, cytochemical reaction was found in the cytoplasm of muscle cells near the postsynaptic membrane, and in a few synaptic vesicles in the vicinity of the active zone. This method may be used for the identification of cholinergic inputs in central and peripheral nerve systems and, generally speaking, for the detection of synaptic activity elicited by specific nerve stimulation.