3 3 diaminobenzidine (dab)

Manufactured by Zhongshan Jinqiao Biotechnology

Sourced in China

DAB is a chromogenic substrate used for visualization in immunohistochemistry and immunocytochemistry applications. It produces a brown precipitate at the site of the target antigen, allowing for the detection and localization of specific proteins in fixed tissue or cell samples.

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Lab products found in correlation

Triton x 100, by MP Biomedicals (3 mentions) Paraformaldehyde (pfa), by Beyotime (3 mentions) Phospho p65, by Cell Signaling Technology (1 mentions) Pvdf membrane, by Merck Group (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) β actin, by Zhongshan Jinqiao Biotechnology (1 mentions) Nf κb p65, by Zhongshan Jinqiao Biotechnology (1 mentions) Bcl 2, by Zhongshan Jinqiao Biotechnology (1 mentions) Neutral gum, by Merck Group (1 mentions) E cadherin, by Proteintech (1 mentions) N cadherin, by Proteintech (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Goat serum, by Zhongshan Jinqiao Biotechnology (1 mentions) Ab201340, by Abcam (1 mentions) Pv 6000, by Zhongshan Jinqiao Biotechnology (1 mentions) Df6280, by Affinity Biosciences (1 mentions) Dapi 8961s, by Cell Signaling Technology (1 mentions)

Spelling variants of this product

Diaminobenzidine (DAB) (4 citations)

9 protocols using 3 3 diaminobenzidine (dab)

Western Blot Analysis of Cell Signaling

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Cells were lysed in RIPA buffer with protease inhibitors and phosphatase inhibitors [15 (link)–16 (link)]. The protein was resolved by SDS/PAGE and blotted on PVDF membranes (Millipore, Bedford, MA, USA)[15 (link)–16 (link)]. The PVDF membranes were incubated with specific primary antibodies at 4°C overnight [15 (link)–16 (link)]. After incubation with HRP-linked secondary antibodies, immunoreactive proteins were visualized using DAB (Beijing Zhongshan Jinqiao biotechnology Co., Ltd.).
Primary antibody against GRα was from Santa Cruz Biotechnology. IκBα, phospho-p65 and cleaved caspase 3 were from Cell Signaling Technology (Beverly, MA, USA). NF-κB p65, Bcl-2, β-actin and HRP-linked secondary antibodies were from Beijing Zhongshan Jinqiao biotechnology Co., Ltd.

He J., Zhou J., Yang W., Zhou Q., Liang X., Pang X., Li J., Pan F, & Liang H. (2017). Dexamethasone affects cell growth/apoptosis/chemosensitivity of colon cancer via glucocorticoid receptor α/NF-κB. Oncotarget, 8(40), 67670-67683.

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Immunohistochemical Evaluation of Protein Markers

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Tissue slices were immersed in 0.01 M citrate buffer (pH 6.0; Wellbio, China). The buffer was brought to a boil and cooked for 20 min before being cooled to room temperature. Tissue slices were washed with PBS (pH 7.2–7.6; China) after cooling. The samples were then incubated at 4°C overnight with diluted primary antibody. Following incubation with primary antibody, tissue slices were washed with PBS, and 50–100 μL of anti-rabbit-IgG antibody-HRP polymer (Thermo Fisher Scientific, China) was added. A working solution of 50–100 μL of the chromogenic reagent DAB (Zhongshan Jinqiao Biotechnology, China) was also added. Tissues were counterstained with hematoxylin before being mounted with neutral gum (Sigma, USA). Tissue slices were then examined under a microscope (OLYMPUS, Japan, BX43). Antibodies for IHC staining included Ki-67 (1:200, Servicebio, China), PGK1 (1:200, Proteintech, China), E-cadherin (1:200, Proteintech, China), and N-cadherin (1:200, Proteintech, China). The Ki-67 expression level was graded based on the percentage of staining. The histochemistry score (H-score) was used to determine the expression levels of PGK1, E-cadherin, and N-cadherin. H-score = ΣPi (i + 1), where i denotes the intensity score and Pi denotes the percentage of the cells.

Jiang Q., Wang Z., Qi Q., Li J., Xin Y, & Qiu J. (2021). lncRNA SNHG26 promoted the growth, metastasis, and cisplatin resistance of tongue squamous cell carcinoma through PGK1/Akt/mTOR signal pathway. Molecular Therapy Oncolytics, 24, 355-370.

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ATDC5 Cell Immunocytochemistry for Col-2

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The transfected ATDC5 were grown on glass coverslips that were previously coated with poly L-lysin.
Then they were xed in 4% paraformaldehyde (Beyotime, China) for 15 min (RT). Permealization was done by immersing slides in 0.3% Triton-X100 (MP Biomedicals, USA) for 15 min. Two drops of the rabbit primary antibody against Col-2 (1:400) were applied to each slide. Then the samples were incubated at 4°C overnight. The next day, the Horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit) was applied to each slide for 1 h. Subsequently, DAB (Zhongshan Jinqiao Biotechnology, China) was prepared and was used for visualizing any antigen-antibody reaction in the cells. The stained sections were observed and recorded by a light microscope (Leica, German).

Zhang K., Li Z., Lu Y., Xiang L., Sun J., & Zhang H. (2021). Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes.

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Spinal Cord Myeloperoxidase Immunostaining

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The 5-µm paraffin-embedded sections, prepared from spinal cord fixed in 4% paraformaldehyde for 24 h at 4˚C, were placed in a pressure cooker with sodium citrate buffer (0.01 mol/l; pH, 6.0) for 20 min at 100˚C for antigen recovery (Wuhan Boster Biological Technology, Ltd.). The activity of endogenous peroxidase was blocked by 3% H₂O₂ at room temperature for 20 min, followed by blocking with 10% goat serum (Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.) for 30 min at room temperature. Primary antibody against myeloperoxidase (1:1,000; cat. no. 14569; Cell Signaling Technology, Inc.) was utilized to incubate the sections overnight at 4˚C and HRP-conjugated secondary antibody (1:1,000; cat. no. ab6721; Abcam) was used to incubate the sections for 1 h at room temperature. After washing with PBS-0.05% Tween-20, DAB (Zhongshan Jinqiao Biotechnology Co., Ltd. China) was used for visualization. Hematoxylin was utilized for nuclei counterstaining at room temperature for 3 min. Finally, images were captured under a light microscope (Leica Microsystems GmbH).

Sun F., Zhang H., Huang T., Shi J., Wei T, & Wang Y. (2022). S100A9 blockade improves the functional recovery after spinal cord injury via mediating neutrophil infiltration. Experimental and Therapeutic Medicine, 23(4), 291.

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Immunohistochemical Analysis of Bax and Bcl-2

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The slides were dewaxed and antigen repaired, then incubated with the primary polyclonal rabbit antibodies of Bax and Bcl-2 (Affinity Biosciences LTD, OH. USA). The concentration of Bax and Bcl-2 was 1:150 and antibodies were incubated for 2 h at 37 °C in a water bath pot (Wiggens Co. LTD, Germany). Biotinylated secondary antibody anti-rabbit IgG (Fuzhou Maixin Biotechnology Development Co. LTD, Fuzhou, China) was used on the sections for 40-min incubation at indoor temperature. Then the slides were colored with DAB (Beijing Zhongshan Jinqiao Biotechnology Co. LTD, Beijing, China). Five sections on each slide were selected randomly for examination under an optical microscope and the Image J was used for quantitative analysis.
A schematic diagram of research plan could be seen in Fig. 1.Fig. 1

A schematic diagram of research plan

Xu H., Xia Y., Qin J., Xu J., Li C, & Wang Y. (2021). Effects of low intensity pulsed ultrasound on expression of B-cell lymphoma-2 and BCL2-Associated X in premature ovarian failure mice induced by 4-vinylcyclohexene diepoxide. Reproductive Biology and Endocrinology : RB&E, 19, 113.

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Immunostaining of Colorectal Cancer Tissues

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The slices of colorectal cancer tissues and para‐carcinoma tissues from patients were deparaffinized with xylene and rehydrated with ethanol. Then, the antigen was unmasked with citrate buffer. Subsequently, the slices were blocked and incubated overnight in primary antibodies against RXRα (cat. no. ab191176), E‐cadherin (cat. no. ab76055), Snail (cat. no. ab53519), vimentin (cat. no. ab92547) and ZEB‐1 (cat. no. ab180905) 4°C, followed by incubation in secondary antibodies from two‐step kits (PV‐6001, PV‐9003; Zhongshan Jinqiao Biotechnology co. LTD). DAB (ZLI‐9017; Zhongshan Jinqiao Biotechnology co. LTD) was used for immunostaining, and nuclei were stained with haematoxylin (AR‐0712; Dingguo Changsheng Biotechnology Co. Ltd). Slices were observed under the Nikon Eclipse 80i fluorescence microscope. ImageJ software was used for analysis.

Lu Z., Liu H., Fu W., Wang Y., Geng J., Wang Y., Yu X., Wang Q., Xu H, & Sui D. (2020). 20(S)‐Protopanaxadiol inhibits epithelial‐mesenchymal transition by promoting retinoid X receptor alpha in human colorectal carcinoma cells. Journal of Cellular and Molecular Medicine, 24(24), 14349-14365.

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Immunohistochemical Analysis of Col-2 in ATDC5 Cells

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The transfected ATDC5 were grown on glass coverslips that were previously coated with poly L-lysin. Then they were xed in 4% paraformaldehyde (Beyotime, China) for 15 min (RT). Permealization was done by immersing slides in 0.3% Triton-X100 (MP Biomedicals, USA) for 15 min. Two drops of the rabbit primary antibody against Col-2 (1:400) were applied to each slide. Then the samples were incubated at 4 °C overnight. The next day, the Horseradish peroxidase-conjugated secondary antibody (goat antirabbit) was applied to each slide for 1 h. Subsequently, DAB (Zhongshan Jinqiao Biotechnology, China) was prepared and was used for visualizing any antigen-antibody reaction in the cells. The stained sections were observed and recorded by a light microscope (Leica, German).

Zhang K., Li Z., Lu Y., Xiang L., Sun J., & Zhang H. (2020). Silencing of Vangl2 Attenuates the Inflammation Promoted by Wnt5a via MAPK and NF-κB Pathway in Chondrocytes.

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Immunocytochemistry of Chondrocytes

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The transfected ATDC5 were grown on glass coverslips that were previously coated with poly L-lysin. Then, they were fixed in 4% paraformaldehyde (Beyotime, China) for 15 min (RT). Permealization was done by immersing slides in 0.3% Triton-X100 (MP Biomedicals, USA) for 15 min. Two drops of the rabbit primary antibody against Col-2 (1:400) were applied to each slide. Then, the samples were incubated at 4 °C overnight. The next day, the Horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit) was applied to each slide for 1 h. Subsequently, DAB (Zhongshan Jinqiao Biotechnology, China) was prepared and was used for visualizing any antigen-antibody reaction in the cells. The stained sections were observed and recorded by a light microscope (Leica, German).

Zhang K., Li Z., Lu Y., Xiang L., Sun J, & Zhang H. (2021). Silencing of Vangl2 attenuates the inflammation promoted by Wnt5a via MAPK and NF-κB pathway in chondrocytes. Journal of Orthopaedic Surgery and Research, 16, 136.

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LDHA Immunostaining in Lung Tissue

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Immunohistochemistry staining was performed using previously reported protocols [37 (link),38 (link)]. The antigen was retrieved using the heat-induced antigen retrieval method, and then 3% hydrogen peroxide was used to quench the endogenous peroxidases. The sections were subsequently incubated with the primary antibody against LDHA (1:100 dilution, DF6280, Affinity) overnight at 4 °C. The following day, the sections were incubated with the secondary antibody (PV-6000, Beijing Zhongshan Jinqiao Bio-technology Co. Ltd, China) at 37 °C for 30 min. Immunoreactivity was visualized using DAB (ZLI-9018; Beijing Zhongshan Jinqiao Biotechnology Co. Ltd.). Cells with brown staining were considered LDHA-positive. Results were visualized by light microscopy. For immunofluorescence staining, paraffin sections of the lung tissue and cell samples were incubated with LDHA, CD68 (ab201340, Abcam)/LDHA, MCP1 (RM01549, ABclonal)/LDHA and Arg-1 (610708, BD)/LDHA overnight at 4 °C. The following day, samples were incubated with the secondary antibody at 37 °C for 40 min. Nuclei were stained with DAPI (8961s; Cell Signaling Technology, Inc., Danvers, MA, USA).

Mao N., Yang H., Yin J., Li Y., Jin F., Li T., Yang X., Sun Y., Liu H., Xu H, & Yang F. (2021). Glycolytic Reprogramming in Silica-Induced Lung Macrophages and Silicosis Reversed by Ac-SDKP Treatment. International Journal of Molecular Sciences, 22(18), 10063.

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