Diaminobenzidine dab substrate kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Diaminobenzidine (DAB) substrate kit is a laboratory reagent used for the detection and visualization of various target molecules in biological samples, such as proteins, enzymes, or other biomolecules. The kit provides the necessary components, including the DAB chromogen, to enable a sensitive and robust colorimetric detection, typically in immunohistochemistry or immunocytochemistry applications.

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Lab products found in correlation

Anti itga2, by Abcam (2 mentions) Signalstain boost ihc anti rabbit hrp reagent, by Cell Signaling Technology (2 mentions) Abc elite, by Vector Laboratories (1 mentions) Anti col1a1, by Merck Group (1 mentions)

Close spelling variants of this product

Metal-enhanced 3,3′-diaminobenzidine (DAB) substrate kit Pierce™ 3,3′ diaminobenzidine (DAB) substrate Kit DAB (3,3’-diaminobenzidine) HRP substrate kit 3,3′-diaminobenzidine (DAB) substrate kit Diaminobenzidine (DAB) kit Diaminobenzidine substrate kit DAB substrate kit Diaminobenzidine (DAB) Diaminobenzidine substrate DAB substrate

4 protocols using diaminobenzidine dab substrate kit

Immunohistochemical Staining Protocol

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The adhered tissues on the slides stored at −20 °C, were first thawed to room temperature. The sections were washed with PBS between all incubations. They were treated with 3% H₂0₂ for 15 min, followed by 0.5% TritonX-100, and normal goat serum. The sections were incubated overnight with primary antibodies.
The slides were washed on day 2 and treated with the corresponding biotinated secondary antibodies, followed by avidin-binding complex (ABC)-elite (Vector; Cat# PK-6100; 1:1000 in PBS; 30 min). The sections were next incubated in a diaminobenzidine (DAB) substrate kit (Cat# 34002, ThermoFisher Scientific) until the color of the tissue turned brown.
The slides were then washed with distilled water, followed by a series of dehydrations and cover slipping.

Soni K.K., Hwang J., Ramalingam M., Kim C., Kim B.C., Jeong H.S, & Jang S. (2023). Endoplasmic Reticulum Stress Causing Apoptosis in a Mouse Model of an Ischemic Spinal Cord Injury. International Journal of Molecular Sciences, 24(2), 1307.

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Western Blot Analysis of E. canis Proteins

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Western blot analysis was performed. Briefly, the protein was subjected to denaturing 12% SDS-PAGE. The proteins were transferred to a nitrocellulose membrane. The membrane was blocked with blocking buffer (5% skim milk in 1% PBS buffer with 0.1% tween 20) at 4 °C overnight and incubated at room temperature for 1–2 h with either the sera of the naturally infected E. canis dogs (1:100) or a rabbit anti-polyhistidine immunoglobulin G (IgG) monoclonal antibody (MoAb) (1:3,000) (Invitrogen™, USA). The membrane was triple washed with the same buffer as listed above. The membrane was then incubated with a goat anti-dog IgM conjugated to horseradish peroxidase, a goat anti-dog IgG conjugated to horseradish peroxidase (1:3,000) (KPL, USA) or goat anti-rabbit IgG/IgA/IgM (H+L) conjugated to horseradish peroxidase (Invitrogen™, USA) for 1 h, washed and developed with a diaminobenzidine (DAB) Substrate Kit (Thermo Scientific, USA) for 3–5 min at room temperature. The specific protein band showed a brown color on the membrane.

Kaewmongkol S., Suwan E., Sirinarumitr T., Jittapalapong S., Fenwick S.G, & Kaewmongkol G. (2020). Detection of specific IgM and IgG antibodies in acute canine monocytic ehrlichiosis that recognize recombinant gp36 antigens. Heliyon, 6(7), e04409.

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Immunohistochemical Analysis of ITGA2

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Formalin-fixed, paraffin-embedded tissue samples were sectioned with a standard microtome at 3- to 5-μm thickness. After deparaffinization and rehydration, heat-induced (98°C) antigen retrieval was performed in a 10 mM sodium citrate buffer (pH 6.0) at a sub-boiling temperature for 10 min. The slides were incubated with hydrogen peroxide 3% (v/v) for 10 min, washed and blocked with 5% FBS in TBST for 1h at room temperature. Next, slides were incubated with primary antibodies anti-ITGA2 (1:500) (Abcam) at 4°C overnight. Primary antibodies were detected using a SignalStain® Boost IHC anti-rabbit HRP Reagent (Cell signaling Technology). The signal was visualized using a diaminobenzidine substrate kit (DAB, Thermo Fisher Scientific) according to the manufacturer’s instructions and nuclei were counterstained with hematoxylin.

Huang Y.L., Liang C.Y., Labitzky V., Ritz D., Oliveira T., Cumin C., Estermann M., Lange T., Everest-Dass A.V, & Jacob F. (2021). Site-specific N-glycosylation of integrin α2 mediates collagen-dependent cell survival. iScience, 24(10), 103168.

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Immunohistochemical Analysis of Collagen and Integrin

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Formalin-fixed, paraffin-embedded tissue samples were sectioned with a standard microtome at 3- to 5 µm thickness. After deparaffinization and rehydration, heat-induced (98°C) antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0) at a sub-boiling temperature for 10 mins. The slides were incubated with hydrogen peroxide 3% (v/v) for 10 min, washed and blocked with 5% FBS in TBST for 1 hr at room temperature. Next, slides were incubated with primary antibodies anti-COL1A1 (1:100) (Sigma-Aldrich) and anti-ITGA2 (1:500) (Abcam) at 4°C overnight. Primary antibodies were detected using a SignalStain Boost IHC anti-rabbit HRP Reagent (Cell signaling Technology). The signal was visualized using a diaminobenzidine substrate kit (DAB, Thermo Fisher Scientific) according to the manufacturer's instructions and nuclei were counterstained with hematoxylin. Immunostaining was scored by the weighted average score (intensity: 0–3, coloring: 0–100% of ITGA2 expression) by two trained scientists independently and discrepancies were resolved by consensus.

Huang Y.L., Liang C.Y., Ritz D., Coelho R., Septiadi D., Estermann M., Cumin C., Rimmer N., Schötzau A., Núñez López M., Fedier A., Konantz M., Vlajnic T., Calabrese D., Lengerke C., David L., Rothen-Rutishauser B., Jacob F, & Heinzelmann-Schwarz V. (2020). Collagen-rich omentum is a premetastatic niche for integrin α2-mediated peritoneal metastasis. eLife, 9, e59442.

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