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3 3 diaminobenzidine (dab)

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DAB is a chromogenic substrate used in immunohistochemistry and immunocytochemistry applications. It produces a brown precipitate at the site of the antigen-antibody reaction, allowing for the visualization and localization of target proteins in tissue sections or cell preparations.

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Lab products found in correlation

Z0331, by Agilent Technologies (3 mentions) Rm 9106 r7, by Thermo Fisher Scientific (3 mentions) Cleaved caspase 3 antibody, by Cell Signaling Technology (2 mentions) Ni u microscope, by Nikon (1 mentions) Scn400 slide scanner, by Leica (1 mentions) Digital image hub, by Leica (1 mentions) Nfatc1 mouse monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) 600 auto biochemical analyzer, by Olympus (1 mentions) Ha 11, by Fortrea (1 mentions) Ab46154, by Abcam (1 mentions) Signalstain boost ihc detection reagent, by Cell Signaling Technology (1 mentions) Ab39256, by Abcam (1 mentions) Ab2350, by Abcam (1 mentions) Iba 1, by Abcam (1 mentions) γh2ax, by Cell Signaling Technology (1 mentions) Harris modified hematoxylin, by Thermo Fisher Scientific (1 mentions) Glial fibrillary acidic protein (gfap), by Cell Signaling Technology (1 mentions) Ihc detection reagent, by Cell Signaling Technology (1 mentions) Permount aqueous mounting media, by Thermo Fisher Scientific (1 mentions) Neuronal nuclei (neun), by Abcam (1 mentions)

Spelling variants of this product

Diaminobenzidine (DAB; (5 citations) 3,3′‐diaminobenzidine tetrahydrochloride (DAB; (2 citations)

20 protocols using 3 3 diaminobenzidine (dab)

1

Histological Evaluation of Liver Fibrosis and Steatosis

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Liver sections from formalin-fixed paraffin-embedded tissue were stained with haematoxylin and eosin (H&E) or picrosirius red to evaluate fibrosis. Fibrosis quantification was performed using Image J (NIH). Optimal cutting temparature compound (OCT)-embedded frozen liver sections were stained with Oil Red O to assess steatosis. For immunohistochemistry, sections were incubated overnight at 4°C with anti-CD68 (Cell Signaling Technology, 97778S), anti-MPO (Abcam, ab9545) or anti-4 hydroxynonenal (4HNE, Abcam, ab46545) primary antibodies. Antibody detection was performed using SignalStain Boost IHC Detection reagent (HRP rabbit, Cell Signaling Technology, 8114S) and immunoreactivity was visualized using 3,3′-diaminobenzidine (DAB) as a substrate (Cell Signaling Technology, 8059S). Nuclear counterstain was performed using haematoxylin. Stained sections were imaged with a 10X objective using a Nikon Ni-U microscope. CD68-and MPO-positive cells were quantified using QuPath (Centre for Cancer Research and Cell Biology, Belfast, UK17 (link)).
Morel C., Chowdhary V., Nagesh P.T., Ribeiro M., Hawryluk D., Catalano D., Adorini L, & Szabo G. (2022). Altered ethanol metabolism and increased oxidative stress enhance alcohol-associated liver injury in farnesoid X receptor-deficient mice. Liver international : official journal of the International Association for the Study of the Liver, 43(1), 100-114.
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2

Immunohistochemical Analysis of Tumor Tissues

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Tumor tissue sections extracted from MMTV-PyMT mice were fixed with 4% (v/v) paraformaldehyde in PBS for 10 min at room temperature. After incubating with primary antibodies overnight at 4°C, endogenous peroxidases in the samples were oxidized by exposure to 0.3% H2O2 in TBS for 15 min at room temperature. Samples were then incubated with secondary antibody, horseradish peroxidase–conjugated anti-rabbit goat antibody (611-103-122, Rockland Immunochemicals, Limerick, PA, USA) at 1:400 diluted in TBS with 10% (v/v) FBS and 10% goat serum for 1 hour at room temperature. Samples were then incubated with 3,3’-Diaminobenzidine (DAB) (Cell Signaling Technology) for signal development and with Mayer’s hematoxylin for counterstaining. After dehydration and mounting, tissue sections were then digitalized using the Leica SCN400 Slide Scanner. Signal intensity and percentage of the signal-positive area were quantified with Leica Digital Image Hub.
Wang W., Hapach L.A., Griggs L., Smart K., Wu Y., Taufalele P.V., Rowe M.M., Young K.M., Bates M.E., Johnson A.C., Ferrell N.J., Pozzi A, & Reinhart-King C.A. (2022). Diabetic hyperglycemia promotes primary tumor progression through glycation-induced tumor extracellular matrix stiffening. Science Advances, 8(46), eabo1673.
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3

Immunocytochemical detection of NFATc1

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Cells were seeded on coverslips and induced to differentiate for 4 days. The cells were then fixed with 4% formaldehyde at room temperature for 20 min and permeabilized in 0.5% tritonX-100 solution for 5 min. To block nonspecific binding, coverslips were incubated in 3% goat serum at room temperature for 30 min and then with NFATc1 mouse monoclonal antibody (1:200, Santa Cruz, sc-7294) overnight at 4°C in PBS with 3% goat serum. After three washes in PBS, the cells were incubated with m-lgGκ BP-HRP antibody (1:1000, Santa Cruz, sc-516102) for 1 h at room temperature and then were counterstained with DAB (Cell signaling Technology).
Zhang D., Lin L., Yang B., Meng Z, & Zhang B. (2020). Knockdown of Tcirg1 inhibits large-osteoclast generation by down-regulating NFATc1 and IP3R2 expression. PLoS ONE, 15(8), e0237354.
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4

Immunohistochemical Analysis of USP46 Expression

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Briefly, 5-µm tumor sections were collected and deparaffinized at 60°C for 10 min followed by 15 min immersion in xylene. The sections were rehydrated via sequential incubation in 100, 90 and 70% ethanol. Samples were rinsed with PBS followed by distilled water and incubated for 30 min in H2O2. Antigen retrieval was performed via microwave irradiation for 10 min. The sections were incubated with an anti-USP46 antibody (1:100; cat. no. ab244215; Abcam) overnight at 4°C. Following primary antibody incubation, the sections were conjugated by SignalStain® Boost IHC Detection reagent (cat. no. 31926S; Cell Signaling Technology, Inc.) at 25°C for 2 h. The sections were then stained with DAB (cat. no. SK-4100; Vector Laboratories, Inc.; Maravai LifeSciences) and mounted with VECTASHIELD® PLUS Antifade mounting medium (cat. no. H-1900; Vector Laboratories, Inc.; Maravai LifeSciences). Sections were visualized using an Olympus 600 auto-biochemical analyzer and a light microscope (Olympus Corporation) at 40× magnification. The percentage of cells with positive staining was determined using ImageJ software (v1.8.0; National Institutes of Health).
Xu L., Zhang B, & Li W. (2021). Downregulated expression levels of USP46 promote the resistance of ovarian cancer to cisplatin and are regulated by PUM2. Molecular Medicine Reports, 23(4), 263.
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5

Comprehensive Immunohistochemical Profiling

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Immunostaining for HA was performed using an anti-HA monoclonal antibody 1:1000 (HA.11, Covance, Berkeley, CA). Analysis of cytokeratin expression was performed using mouse cytokeratin-pan-antibody-cocktail (MA5-13203, Thermo Scientific). Analysis of S100 expression was performed using rabbit polyclonal antibody Z0331 (1:400) for S100 (Dako; Glostrup, Denmark). Detection of MAPK activation was performed using a 1:100 dilution of an antibody to phospho-ERK (4370, Cell Signaling). Cell proliferation was detected using a 1:250 dilution of a rabbit monoclonal antibody to Ki67 (RM-9106-R7 Thermo Fisher Scientific). Detection of HRP activity was performed using DAB (Cell Signaling). Sections were counterstained with hematoxylin.
Grigore F., Yang H., Hanson N.D., VanBrocklin M.W., Sarver A.L, & Robinson J.P. (2020). BRAF inhibition in melanoma is associated with the dysregulation of histone methylation and histone methyltransferases. Neoplasia (New York, N.Y.), 22(9), 376-389.
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6

Tissue Immunohistochemistry Protocol

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Tissue sections were deparaffinized in xylene and rehydrated with gradient alcohol, followed by antigen repair using citrate buffer. The serum-blocked sections were then incubated with the required primary antibody dilutions, including TRAF6 (1:50), phospho-P65 (1:200), and phosphor-ERK (1:200), overnight at 4 °C. Thereafter, the sections were sequentially incubated with biotin-labeled secondary antibodies of goat anti-rabbit IgG and streptavidin-conjugated peroxidase for 30 min at 37 °C. The chromogenic reaction was induced using DAB (Cell Signaling Technology, Danvers, MA, USA) under a microscope. ImageJ software was utilized to assess positively stained cells in random regions.
Peng X., Wang T., Wang Q., Zhao Y., Xu H., Yang H., Gu Y., Tao Y., Yan B., Xu Y, & Geng D. (2024). Pan-histone deacetylase inhibitor vorinostat suppresses osteoclastic bone resorption through modulation of RANKL-evoked signaling and ameliorates ovariectomy-induced bone loss. Cell Communication and Signaling : CCS, 22, 160.
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7

Immunohistochemical Analysis of Cellular Markers

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Analysis of S100 expression was performed using rabbit polyclonal antibody Z0331 (1:400) for S100 (Dako; Glostrup, Denmark). Detection of MAPK activation was performed using a 1:100 dilution of an antibody to phospho-ERK (4370, Cell Signaling). Cell proliferation was detected using a 1:250 dilution of a rabbit monoclonal antibody to Ki67 (RM-9106-R7 Thermo Scientific, Waltham, MA). IHC HA was performed using a 1:200) dilution of the HA.11 antibody. Apoptosis detection was performed using a 1:250 dilution of Cleaved Caspase-3 antibody (9579 Cell Signaling). Controls were carried out without primary antibody on corresponding sections. Detection of HRP activity was performed using DAB (Cell Signaling). Sections were counterstained with hematoxylin.
Yang H., Kircher D.A., Kim K.H., Grossmann A.H., VanBrocklin M.W., Holmen S.L, & Robinson J.P. (2017). Activated MEK cooperates with Cdkn2a and Pten loss to promote the development and maintenance of melanoma. Oncogene, 36(27), 3842-3851.
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8

Ovarian VEGF Immunohistochemical Analysis

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Immunohistochemical analysis was performed according to a well-established method ( 20 (link) ). Frozen ovarian tissue sections were air-dried for 30 min at room temperature. These sections were washed with PBS twice for 5 min each. The sections were treated with 3% hydrogen peroxidase in methanol to quench endogenous peroxidase activity and subsequently washed with PBS. Ovarian tissue sections were incubated with a blocking solution for 7 min at room temperature in a humidified chamber. These sections were incubated with VEGF (Abcam, ab46154, dilution; 1/50), VEGFR1 (Abcam, ab2350, dilution; 1/25) and VEGFR2 (Abcam, ab39256, dilution; 1/50) antibodies at +4°C overnight. The following day, the sections were incubated with SignalStain Boost IHC Detection Reagent (Cell Signaling, 8114). The reaction products were visualized using Dab (Cell Signaling, #8059). These sections were counterstained with Mayer’s haematoxylin and mounted with Entellan. Then, images were taken with a light microscope.
Kandemir Y.B., Konuk E., Katırcı E., Xxx F, & Behram M. (2019). Is the effect of melatonin on vascular endothelial growth factor receptor-2 associated with angiogenesis in the rat ovary?. Clinics, 74, e658.
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9

Immunohistochemistry for Brain Tissue Analysis

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Immunohistochemistry was performed using standard protocols. Briefly, brains were explanted, fixed in formalin for 24 h and embedded in paraffin. Sections (4 μm) were cut, baked for 1 h at 65°C, de-waxed in 2 successive xylene baths of 5 min and hydrated for 5 min using an alcohol gradient (ethanol 100%, 90%, 70%, 50%, 25%). The slides were incubated in 3% hydrogen peroxide/methanol solution for 10 min. Antigen retrieval was performed using citrate buffer (10 mM sodium citrate, 0.05% Tween™ 20, pH 6) with heating to 95°C in a steamer for 25 min. After cooling down to room temperature, the slides were blocked and incubated overnight at 4°C with a primary antibody against GFAP (1:50; Cell Signaling Technology®, Danvers, MA), IBA1 (1:8,000; Abcam®, Cambridge, MA), NeuN (1:3,500; Abcam) or γ-H2AX (1:1,000; Cell Signaling Technology). The slides were then incubated with ready-to-use IHC detection reagent (100 μl; Cell Signaling Technology) at room temperature for 1 h, rinsed, and incubated with DAB (Cell Signaling Technology) for 3–5 min. Tissues were counterstained or not with Harris modified Hematoxylin (Fisher Scientific™, Waltham, MA) for 30 s, dehydrated via an alcohol gradient (ethanol 25%, 50%, 70%, 90%, 100%) and soaked twice in xylene. A drop of Permount™ aqueous mounting media (Fisher Scientific) was added on the top of the section and covered.
Saki M., Bhat K., Sodhi S.S., Nguyen N.T., Kornblum H.I, & Pajonk F. (2019). Effects of Brain Irradiation in Immune-Competent and Immune-Compromised Mouse Models. Radiation research, 193(2), 186-194.
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10

Immunohistochemical Analysis of S100, MAPK, and Apoptosis

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Analysis of S100 expression was performed using a rabbit polyclonal antibody Z0331 (1:400) for S100 (Dako; Glostrup, Denmark). Detection of MAPK activation was performed using a 1:100 dilution of an antibody against phospho-ERK (4370, Cell Signaling). Cell proliferation was detected using a 1:250 dilution of a rabbit monoclonal antibody against Ki67 (RM-9106-R7 Thermo Scientific, Waltham, MA, USA). IHC HA was performed using a 1:200) dilution of the HA.11 antibody. Apoptosis detection was performed using a 1:250 dilution of the cleaved caspase-3 antibody (9579 Cell Signaling). Controls were conducted without primary antibody on corresponding sections. Detection of HRP activity was performed using DAB (Cell Signaling). Sections were counterstained with hematoxylin.
Yang H., Kircher D.A., Kim K.H., Grossmann A.H., VanBrocklin M.W., Holmen S.L, & Robinson J.P. (2017). Activated MEK cooperates with Cdkn2a and Pten loss to promote the development and maintenance of melanoma. Oncogene, 36(27), 3842-3851.
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