3 3 diaminobenzidine dab liquid substrate system

Manufactured by Merck Group

Sourced in United States

3,3-diaminobenzidine (DAB) liquid substrate system is a lab equipment product used for the detection and visualization of enzymes in immunohistochemistry and immunocytochemistry applications. It provides a chromogenic substrate for the localization of peroxidase-conjugated antibodies or other peroxidase-labeled reagents.

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5 protocols using 3 3 diaminobenzidine dab liquid substrate system

Immunohistochemical Analysis of C1GALT1 Expression

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Paraffin-embedded tissue sections were incubated with an anti-C1GALT1 (1:300) antibody overnight at 4°C. Super Sensitive link-Label IHC detection System (BioGenex, California, CA) was used and signals were visualized with 3,3-diaminobenzidine (DAB) liquid substrate system (Sigma, St. Louis, MO). All sections were counterstained with hematoxylin. Negative controls were performed by replacing primary antibody with a control IgG at the same concentration. The staining intensity and positive ratio of C1GALT1 were observed under microscope by two independent scorers blinded to the clinical parameters.

Hung J.S., Huang J., Lin Y.C., Huang M.J., Lee P.H., Lai H.S., Liang J.T, & Huang M.C. (2014). C1GALT1 overexpression promotes the invasive behavior of colon cancer cells through modifying O-glycosylation of FGFR2. Oncotarget, 5(8), 2096-2106.

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Biochemical Assays for Neurological Disorders

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Acetylthiocholine iodide, adenine, ketamine, xylazine, thiobarbituric acid, 1,1,3,3-tetramethoxypropane, 3,3-diaminobenzidine (DAB) liquid substrate system, hydrogen peroxide (H₂O₂), Sirius red and poly-L-lysine were obtained from Sigma-Aldrich Co., USA. Potassium dichromate, silver nitrate and formaldehyde were obtained from HiMedia laboratories Pvt. Ltd., India. Acetic acid, cresyl violet, Nitro blue tetrazolium (NBT), 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB), cytochrome c, pyrogallol, salicylic acid, tri-sodium citrate, copper sulphate, potassium ferricyanide, paraformaldehyde, picric acid, Triton X-100, sodium dodecyl sulphate and other chemicals were of analytical reagent grade, procured from SISCO Research Laboratories Pvt. Ltd., India. Rabbit anti-Glial fibrillary acidic protein (GFAP) antibody (ab7260) and rabbit anti-tyrosine hydroxylase (TH) antibody (ab112) were procured from Abcam (Cambridge, UK). Anti-rabbit goat secondary antibody conjugated with horseradish peroxidase (HRP; ap307p) was procured from Millipore Co. (USA). Urea, creatinine and uric acid assay kits were obtained from Coral Clinical Systems, Goa, India (Reference no. 1102240075, 1101070275, 1102260275).

Mazumder M.K., Paul R., Bhattacharya P, & Borah A. (2019). Neurological sequel of chronic kidney disease: From diminished Acetylcholinesterase activity to mitochondrial dysfunctions, oxidative stress and inflammation in mice brain. Scientific Reports, 9, 3097.

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Cardiac Protein Expression Evaluation

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After 21 days of cell cultivation, the cells cultured on coverslips (Thermo Scientific, UK) from the basal and cardiogenic induced groups (n=5) were used to evaluate the expression of cardiac specific proteins. After fixation for 30 min at 4°C with 4% paraformaldehyde, the cells were blocked in 10% AB-serum in 1% BSA in PBS for 30 min at 4°C, and then incubated with rabbit monoclonal primary antibody against human connexin43 (Cx43) (Sigma-aldrich, St. Louis, MO, USA) for 2 h at 4°C. After being washed with PBS, the cells were incubated with mouse anti-rabbit peroxidase-conjugated secondary antibody (Immuno Tools GmbH, Friesoythe, Germany) for 60 min at 37°C. Finally, the immunoreaction was detected by using 3,3′-Diaminobenzidine (DAB) liquid substrate system (Sigma-aldrich, St. Louis, MO, USA) and analyzed under the Axiostar plus light microscope (Carl Zeiss, Germany). Photographs were taken with Canon pc 1049 PowershotG5 (Canon, USA) and were used to determine and normalize the average level of color intensity in both the basal and cardiogenic induced groups using iSolution FL Auto ×64.

Markmee R., Aungsuchawan S., Narakornsak S., Tancharoen W., Bumrungkit K., Pangchaidee N., Pothacharoen P, & Puaninta C. (2017). Differentiation of mesenchymal stem cells from human amniotic fluid to cardiomyocyte-like cells. Molecular Medicine Reports, 16(5), 6068-6076.

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GALNT2 Protein Expression Analysis

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The paraffin‐embedded tissue section was incubated with an anti‐GALNT2 antibody (1 : 100, Sigma, MO, USA) at 4 °C for 16 h. Super Sensitive™ Link‐Label IHC Detection System (BioGenex, Fremont, CA, USA) was used and signals were visualized through a 3,3‐diaminobenzidine (DAB) liquid substrate system (Sigma, St. Louis, MO, USA). The tissues were counterstained with hematoxylin and mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed by replacing the primary antibody with a control IgG at the same concentration. The intensity of immunohistochemical staining was scored by two independent researchers who were blinded to the clinical data of patients. The scores were categorized into four groups as follows: 0 (negative), 1 (weakly positive), 2 (moderately positive), and 3 (strongly positive).

Liao Y., Chuang Y., Lin H., Lin N., Hsu T., Hsieh S., Chen S., Hung J., Yang H., Liang J., Huang M, & Huang J. (2022). GALNT2 promotes invasiveness of colorectal cancer cells partly through AXL. Molecular Oncology, 17(1), 119-133.

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Immunohistochemical Analysis of C1GALT1 in Gastric Adenocarcinoma

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Tissue microarray of gastric adenocarcinoma from 111 patients was purchased (HStm-Ade178Sur-01, US Biomax, Inc., MD, USA) for immunohistochemical staining. The tissue microarray was incubated with an anti-C1GALT1 antibody (1:100, Santa Cruz Biotechnology, CA, USA) at 4 °C for 16 h. Super Sensitive^TM Link-Label IHC Detection System (BioGenex, CA, USA) was used and signals were visualized through a 3,3-diaminobenzidine (DAB) liquid substrate system (Sigma, MO, USA). The tissues were counterstained with hematoxylin and mounted with UltraKitt (J.T. Baker, Deventer, Holland). Negative controls were performed through replacing the primary antibody with a control IgG at the same concentration.

Lee P.C., Chen S.T., Kuo T.C., Lin T.C., Lin M.C., Huang J., Hung J.S., Hsu C.L., Juan H.F., Lee P.H, & Huang M.C. (2020). C1GALT1 is associated with poor survival and promotes soluble Ephrin A1-mediated cell migration through activation of EPHA2 in gastric cancer. Oncogene, 39(13), 2724-2740.

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