Immunohistochemistry was performed using a power vision two-step kit (ZhongShan Golden Bridge Biotechnology, China). Paraffin-embedded fixed tissue sections (5 μm) were deparaffinized, rehydrated, and pretreated for antigen retrieval in Tris/EDTA. The sections were incubated with anti-E-cadherin rabbit polyclonal antibody and anti-α-smooth muscle actin (α-SMA) mouse monoclonal antibody (Santa Cruz Biotechnology, USA) at 4°C overnight, with the secondary antibody at room temperature for 30 min. Positive immunoreaction was identified after incubation with 3,3-diaminobenzidine tetrahydrochloride (ZhongShan Golden Bridge Biotechnology) and counterstaining with hematoxylin. Negative controls without primary antibodies were carefully examined for each reaction. Sections were photographed with an upright fluorescence microscopic imaging system (BX51 Olympus, Japan). To analyze the expression of E-cadherin and α-SMA in glomeruli and interstitium, 10 nonoverlapping high-power fields (magnification 400×) were randomly selected from each kidney section. Data are reported as the mean optical density in each field area.
3 3 diaminobenzidine tetrahydrochloride
Manufactured by Zhongshan Golden Bridge Biotechnology
3,3-diaminobenzidine tetrahydrochloride (DAB) is a chromogenic substrate used in immunohistochemistry and other biochemical applications. It is a brown-colored precipitate that forms in the presence of peroxidase enzymes, allowing for the visualization and localization of target antigens or proteins in biological samples.
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2 protocols using 3 3 diaminobenzidine tetrahydrochloride
1
Immunohistochemical Analysis of E-cadherin and α-SMA in Kidney Tissue
2
Immunohistochemical Analysis of HMGB1 Expression
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After the electrophysiology studies, the brain was rapidly removed, and samples were immersed in 10% formalin for 24 h, embedded in paraffin and cut into 5-μm thick slices. The paraffin sections were deparaffinized, rehydrated and soaked in 0.1 M of citric acid buffer for 15 min at 92–98°C in a microwave oven, and washed with PBS. Then, the sections were incubated with the primary antibodies of anti-HMGB1 (ab172730-Rabbit monoclonal IgG, 1:5,000; Abcam) overnight at 4°C. Subsequently, the samples were incubated with horseradish peroxidase-conjugated secondary antibodies of rabbit anti-sheep IgG (KPL, Gaithersburg, MD, USA) and goat anti-mouse IgG (Zhongshan Golden Bridge Biotechnology) for 1 h at 37°C. Immunore-activity was developed with 3,3′-diaminobenzidine tetrahydrochloride (Zhongshan Golden Bridge Biotechnology). Finally, the sections were counter-stained with hematoxylin, mounted and examined by microscopy.
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