IHC staining was performed using the Histostain-Plus Bulk Kit (Invitrogen, Camarillo, CA, USA). Briefly, after deparaffinization, the sections were microwave-treated in citrate buffer (pH 6.0) (Vector Laboratories, Burlingame, CA, USA) for heat-induced epitope retrieval and incubated in 3% hydrogen peroxide to inhibit endogenous peroxidases. The sections were incubated overnight at 4°C with primary mouse anti-human p65 monoclonal antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Bound antibodies were visualized using a 3,3′-diaminobenzidine detection kit (Vector Laboratories) and slides counterstained with hematoxylin. Each tissue slice was photographed 5 times at 400× magnification. The presence of brown-yellowish granules in the cell nucleus was the criterion used to classify cells as p65-positive. Total cell number and number of p65-positive cells in epithelia, glands, and submucosa were determined by 2 independent researchers and the means were calculated. The p65-positive cell ratio was calculated according to the following formula and used as a parameter of NF-κB activity:
3 3 diaminobenzidine detection kit
Manufactured by Vector Laboratories
Sourced in United States
The 3,3'-diaminobenzidine detection kit is a laboratory product used for the visualization and detection of specific target molecules in biological samples. It provides a sensitive and reliable method for identifying the presence and distribution of these target molecules within the sample.
Automatically generated - may contain errors
Lab products found in correlation
Histostain plus bulk kit, by Thermo Fisher Scientific (1 mentions)
Citrate buffer, by Vector Laboratories (1 mentions)
Evos fl auto, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit igg, by Vector Laboratories (1 mentions)
Alexa fluor 647 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Sc 28385, by Santa Cruz Biotechnology (1 mentions)
3 protocols using 3 3 diaminobenzidine detection kit
1
IHC-Based Quantification of NF-κB Activation
2
Immunodetection of NHE8 Protein
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Liver tissue sections (4–5 μm thick) and cultured human hepatocyte cells (HepG2) were used to detect the expression of NHE8 protein. The NHE8 antibody was used at a dilution of 1:200.2 (link) For tissue samples, a goat anti-rabbit IgG (dilution 1:500; catalog no. SK-4105; Vector Laboratories, Inc, Burlingame, CA) was used as the secondary antibody, and a 3,3′-diaminobenzidine detection kit (catalog no. SK-4105; Vector Laboratories, Inc, Burlingame, CA) was used for signal detection. For cell samples, Alexa Fluor 647 goat anti-rabbit IgG (dilution 1:400; catalog No. A27040, Thermo Fisher Scientific) was used as the secondary antibody. Stained tissue sections and cells were then observed under microscope (EVOS FL Auto, Thermo Fisher Scientific).
3
Immunohistochemical Analysis of ANXA2 in Prostate Cancer
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Paraffin-embedded PCa tissue sections were obtained from the SDM College of Medical Sciences and Hospital, Dharwad in accordance with established core protocols and Institutional Ethical Board approval. ANXA2 protein expression in tissues collected from PCa patients, including hormone-dependent, metastatic malignant tissue and adjacent non-malignant epithelium, and also in mice tissues from animal experiments, was determined by immuno-histochemical staining as described previously19 (link). First, tissue sections were stained with hematoxylin–eosin to assess the histological form and grade of tumors and then subjected to immunohistochemistry. In brief, following deparaffinization and endogenous peroxidase blockage, the sections were heated in 0.01 M citrate buffer solution (pH 6.0) in a water bath at 98 °C for 20 min; then incubated with 1:100 diluted monoclonal anti-body to ANXA2 (sc-28385, Santa Cruz Biotechnology, TX, USA) overnight at 4 °C; and visualized using a 3,3′-diaminobenzidine detection kit (Vector labs). Staining intensity of ANXA2 was graded by microscopic observation on a scale of 0 to 3+ , where 0 and 3+ indicates no staining and strong staining, respectively.
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