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.
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.
Automatically generated - may contain errors
Lab products found in correlation
Inverted microscope, by Zeiss (2 mentions)
Signalstain boost ihc detection reagent, by Cell Signaling Technology (1 mentions)
Biotinylated goat anti mouse igg, by Vector Laboratories (1 mentions)
Vectastain abc reagent, by Vector Laboratories (1 mentions)
Meyer s hematoxylin, by Vector Laboratories (1 mentions)
Mouse anti vcam 1 monoclonal antibody, by Abcam (1 mentions)
Bx51 microscope, by Olympus (1 mentions)
3 protocols using 3 3 diaminobenzidine tetrahydrochloride solution
1
Immunohistochemical Analysis of p53 Expression
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.
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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