3 3 diaminobenzidine tetrahydrochloride dab substrate

Manufactured by Thermo Fisher Scientific

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3,3'-diaminobenzidine tetrahydrochloride (DAB) substrate is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications. It produces a brown, insoluble precipitate upon oxidation by peroxidase enzymes, allowing for the visualization of target antigens in biological samples.

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Lab products found in correlation

Immpress anti mouse igg, by Vector Laboratories (1 mentions) Aperio cs2, by Leica (1 mentions) Interleukin 6 (il 6), by Abcam (1 mentions) Tnf α, by Abcam (1 mentions) Hematoxylin, by Agilent Technologies (1 mentions) Anti adenovirus type 5 antibody, by Abcam (1 mentions) Digital sight ds fi1, by Nikon (1 mentions) Vectastain elite abc kit, by Vector Laboratories (1 mentions) Biotinylated goat anti rabbit secondary antibody, by Vector Laboratories (1 mentions)

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DAB (142 citations) DAB substrate (44 citations) Diaminobenzidine (DAB) (29 citations) 3,3′-diaminobenzidine substrate (10 citations) 3,3-diaminobenzidine tetrahydrochloride (DAB) (8 citations) 3,3′-diaminobenzidine tetrahydrochloride substrate (3 citations) 3,3′-Diaminobenzidine (DAB; (3 citations) 3,3′-diaminobenzidine tetrahydrochloride (2 citations) Diaminobenzidine substrate (2 citations)

5 protocols using 3 3 diaminobenzidine tetrahydrochloride dab substrate

Serological Analyses of RSV Antibodies

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RSV F protein-specific antibodies (IgG, IgG1, and IgG2a) were determined in samples by enzyme-linked immunosorbent assay (ELISA) as previously described ^{20 (link)}. To determine hemagglutination inhibition (HI) titers, serum samples were incubated with receptor destroying enzyme (RDE, Denka Seiken, Japan) and heated at 56 °C. In brief, HI titers were determined using inactivated PR8 virus and 1% chicken erythrocyte suspension with two-fold diluted serum samples after RDE treatment.
RSV-specific neutralizing antibody titers in immune sera were evaluated by a standard method as previously described ^{21 (link)}. Briefly, the serum samples were heat-inactivated at 56°C and serially diluted two-fold in serum-free DMEM. Equal volumes of RSV (300 PFU/well) were mixed with diluted sera. A mixture of RSV with or without immune sera was incubated at 33°C, 5% CO₂ for 1 h prior to incubation in the HEp-2 cell monolayers. The next steps were followed by an immune-plaque assay procedure as described previously ^{15 (link)}. After fixing with 5% formaldehyde in PBS and blocked with 5% non-fat dry milk in PBST, anti-RSV F monoclonal antibody (131-2A, Millipore) and then HRP conjugated anti-mouse IgG antibody were used. Individual plaques were developed using 3,3’-diaminobenzidine tetrahydrochloride (DAB) substrate (Invitrogen, Camarillo, CA) and then counted.

Lee Y.N., Hwang H.S., Kim M.C., Lee Y.T., Kim Y.J., Lee F.E, & Kang S.M. (2015). Protection against Respiratory Syncytial Virus by Inactivated Influenza Virus Carrying a Fusion Protein Neutralizing Epitope in a chimeric hemagglutinin. Nanomedicine : nanotechnology, biology, and medicine, 12(3), 759-770.

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TCID50 Assay for Virus Quantification

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Next, 6 × 10³ cells/well were seeded into a 0.1% gelatin-coated 96-well plate. Cells were infected with 50 µL of six serial dilutions of virus-containing media, ranging from undiluted to 10⁻⁵ dilutions. Then, 72 h post infection, cells were fixed and permeabilized with 100% methanol for 30 min at −20 °C, washed with PBS followed by PBS-T, and blocked with 1% BSA and 0.2% skim milk in PBS-T. Afterwards, 3% hydrogen peroxide was added to block endogenous peroxidase activity. Cells were stained with anti-NS5A (1:25,000), ImmPRESS anti-mouse IgG (1:3000) (Vector Laboratories), and the 3,3’-diaminobenzidine tetrahydrochloride (DAB) substrate (1 drop/mL) (Invitrogen), respectively. NS5A-positive wells were counted and recorded using a light microscope. TCID50 was calculated with a Reed & Muench Calculator, as previously described [63 (link)].

Liu D., Ndongwe T.P., Ji J., Huber A.D., Michailidis E., Rice C.M., Ralston R., Tedbury P.R, & Sarafianos S.G. (2023). Mechanisms of Action of the Host-Targeting Agent Cyclosporin A and Direct-Acting Antiviral Agents against Hepatitis C Virus. Viruses, 15(4), 981.

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Immunohistochemical Detection of Inflammatory Markers

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General immunohistochemical staining method was applied, briefly, paraffinembedded sections were de-paraffinized, rinsed with PBS repeatedly, and then blocked with 5% serum for 0.5 h. Sections were then stained with primary antibodies against interleukin-1 (IL-1, Abcam, Cambridge, UK), tumor necrosis factor-a (TNF-α, Abcam Inc., Cambridge, MA, USA), or interleukin-6 (IL-6, Abcam, Cambridge, UK) overnight at 4 °C. The sections were treated and incubated after washing by PBS at 37 °C for 0.5 h. Subsequently, the sections were detected with 3,3-diaminobenzidine tetrahydrochloride (DAB) substrate (Invitrogen, Carlsbad, CA, USA) and counterstained with hematoxylin. The samples were detected using a digital pathology system (Aperio CS2, Leica, Germany).

Li W., Yu Q., Yao H., Zhu Y., Topham P.D., Yue K., Ren L, & Wang L. (2019). Superhydrophobic hierarchical fiber/bead composite membranes for efficient treatment of burns. Acta biomaterialia, 92.

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Immunohistochemical Detection of Adenovirus

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Tumor tissues of two mice per group were obtained by necropsy at 7 days after the second intravenous injection. All tissues were fixed for at least 24 h in 10% formaldehyde, embedded in paraffin, and then cut into 40-μm sections. Tissue section slides were deparaffinized with xylene twice for 10 min, and the slides were rehydrated using a graded alcohol series. Following antigen retrieval, endogenous peroxidase from each tissue section was removed using 3% hydrogen peroxide. After blocking for 1 h with 5% BSA, slides were incubated with anti-adenovirus type 5 antibody (Abcam, UK) overnight at 4°C to detect adenovirus structural proteins (e.g., hexon, penton, and fiber). Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG was used as a secondary antibody and incubated for 1 h at room temperature. To detect the chromogenic activity of HRP, a mixture of 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate (Thermo Fisher Scientific, 34002) was added for 5–10 min. After washing with distilled water, hematoxylin (Dako, Denmark) was added for 2–5 min to stain the nuclei. The dehydrated and cleared tissue sections were mounted with mounting media (xylene:mount = 1:1) for microscopy.

Choi S., Hong J.A., Choi H.J, & Song J.J. (2022). Enhanced tumor targeting and timely viral release of mesenchymal stem cells/oncolytic virus complex due to GRP78 and inducible E1B55K expressions greatly increase the antitumor effect of systemic treatment. Molecular Therapy Oncolytics, 27, 26-47.

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Immunohistochemical Analysis of Sinonasal Tissue

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Freshly harvested sinonasal tissue from a minimum of three different subjects was fixed in formalin, embedded in paraffin and routinely processed. Sections were then deparafinnized in graded ethanol baths followed by antigen retrieval with boiling citrate buffer (pH 6.6) for 10 minutes. Tissue endogenous peroxidases were quenched with 0.3% hydrogen peroxide and slides were then incubated with 3% BSA in PBS to prevent nonspecific binding. Slides were then incubated with primary rabbit anti-human A20/TNFAIP3, mouse anti-human Cezanne, or rabbit anti-human CYLD antibody (Abcam; Cambridge, UK) for 1h at room temperature, and then a biotinylated goat anti-rabbit secondary antibody (Vector Laboratories, Burlingame, CA) for 1 h. Signal detection was performed using an avidin-peroxidase detection agent (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA) and 3,3′-diaminobenzidine tetrahydrochloride (DAB) substrate (Thermo Scientific, Waltham, MA). The sections were then counterstained with hematoxylin for 10 min, mounted, and subsequently analyzed with a Nikon Eclipse Ti inverted microscope and a Nikon Digital Sight DS-Fi1 color camera.

Li P., Wang Y, & Turner J.H. (2015). Pro-inflammatory Mediators Alter Expression of NF-Kappa B-Regulating Deubiquitinases in Sinonasal Epithelial Cells. International forum of allergy & rhinology, 5(7), 583-589.

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