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3 3 diaminobenzidine tetrahydrochloride solution

Manufactured by Vector Laboratories

3,3'-diaminobenzidine tetrahydrochloride solution is a chemical compound used as a chromogenic substrate in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It undergoes an enzymatic reaction, resulting in the formation of a brown precipitate that can be visualized to detect the presence of specific target proteins or antigens in biological samples.

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3 protocols using 3 3 diaminobenzidine tetrahydrochloride solution

1

Immunohistochemical Analysis of p53 Expression

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Dissected tissues were fixed in formalin, embedded in paraffin, and sectioned into 3 μm slices by standard procedures. Sections were deparaffinized/rehydrated and stained with hematoxylin–eosin. For antigen retrieval, sections were autoclaved for 15 min in 10 mM sodium citrate buffer (pH 6·0). Tissue sections were incubated with rabbit monoclonal anti-p53 antibody (clone 7F5, 1/160 dilution; Cell Signaling Technology, Cat# 2527; RRID: AB_331211) at 4 °C overnight, followed by incubation with SignalStain Boost IHC Detection Reagent (Cell Signaling Technology, Cat# 8114) for 30 min at room temperature. Immunoreactive signals were visualized with 3,3′-diaminobenzidine tetrahydrochloride solution (Vector, Cat# SK-4105), and nuclei were counterstained with hematoxylin.
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2

Detecting PEDV Viral Proteins in Vero Cells

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To detect expression of viral proteins, KPEDV-9 infected Vero cells were fixed with 5% PFA for 5 min and permeabilized with 1% NP40. Following three washes with PBS, cells were incubated with 1:5000 dilution of mouse anti-PEDV polyclonal antibody for 1 h. Cells were washed three times with PBS and then incubated with 1:1000 dilution of goat anti-mouse IgG conjugated with horseradish peroxidase. KPEDV-9 infected Vero cells were stained using a 3, 3′-diaminobenzidine tetrahydrochloride solution containing NiCl2 and H2O2 (Vector Laboratories). Clusters of immunostained cells were observed under the inverted microscope (Zeiss) and were presented as the ratio between mock-treated and DMSO treated cells.
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3

Quantitative Immunohistochemistry of VCAM-1 in Lung

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Immunohistochemistry was performed as described previously with minor modifications [23 (link)]. Briefly, tissue slides printed with normal lung or lung cancer tissues were purchased from SuperBioChips Laboratories (Seoul, Korea). The slides were incubated first with mouse anti-VCAM-1 monoclonal antibody (1:200; Abcam, Cambridge, MA, USA) and then with biotinylated goat anti-mouse IgG (1:200; Vector Laboratories, Burlingame, CA, USA). Immunoreactive proteins were visualized using VECTASTAIN ABC Reagent (Vector Laboratories). For chromogenic reactions, the slides were incubated with fresh 3,3′-diaminobenzidine tetrahydrochloride solution (Vector Laboratories). All samples were counterstained with Meyer’s hematoxylin (Vector Laboratories). VCAM-1 expression was observed by light microscopy using an Olympus BX51 microscope (Olympus, Tokyo, Japan), and RGB images were acquired using Paint Shop Pro X software (Corel, Ottawa, ON, Canada). After performing background subtraction, VCAM-1 expression was quantified by measuring the signal density with Image J software version 1.48v (National Institutes of Health, Bethesda, MA, USA).
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