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Dab chromogen

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DAB chromogen is a laboratory reagent used as a substrate for the visualization of enzyme-based immunohistochemistry and in situ hybridization techniques. It produces a brown colored precipitate at the site of the enzyme-substrate reaction, allowing for the detection and localization of target molecules in biological samples.

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Lab products found in correlation

Rabbit anti mouse cleaved caspase 3 antibody, by Cell Signaling Technology (1 mentions) Ab190966, by Abcam (1 mentions) Alexa 555, by Thermo Fisher Scientific (1 mentions) Sc 101199, by Santa Cruz Biotechnology (1 mentions) Alexa 488, by Thermo Fisher Scientific (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) Ab955, by Abcam (1 mentions) Anti α sma, by Santa Cruz Biotechnology (1 mentions) Ds u3, by Nikon (1 mentions) Anti collagen 1, by Santa Cruz Biotechnology (1 mentions) Tris cl, by Wisent (1 mentions) Sodium chloride (nacl), by Thermo Fisher Scientific (1 mentions) Sodium dodecyl sulfate (sds), by Roche (1 mentions) Mgcl2, by Thermo Fisher Scientific (1 mentions) Ethylene diamine tetraacetic acid (edta), by Merck Group (1 mentions) Proteinase k, by Qiagen (1 mentions) Chondroitinase abc, by Merck Group (1 mentions)

Close spelling variants of this product

Diaminobenzidine (DAB) chromogen

6 protocols using dab chromogen

1

Quantification of Apoptosis and T-cell Infiltration in Tumor Sections

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Tumor sections (three for each tumor) were stained with hematoxylin-eosin (HE) as described (36 (link)). HE stained tumor tissues were examined with a microscope at 400 × magnification.
Tumor sections (five for each tumor) were immunostained with antibodies for cleaved caspase-3 and CD3 expression. Rabbit anti-mouse cleaved caspase-3 antibody and rabbit anti-mouse CD3ε antibody (1:200 dilution) were purchased from Cell Signaling Technology (Danvers, MA, USA). The sections were treated with antigen retrieval solution followed by 3% hydrogen peroxide treatment, blocked with 5% goat serum, incubated with primary antibody and with biotin-labeled secondary antibody (goat anti-rabbit; 1:1,000 dilution; Abcam, Cambridge, UK). The stained tissues were visualized by using the DAB chromogen (Abcam) reagent and counterstained with hematoxylin. For quantification of immunohistochemical staining, positive cell percentages were counted in five random 400 × microscopic fields for each tissue section.
Li P., Yang L., Li T., Bin S., Sun B., Huang Y., Yang K., Shan D., Gu H, & Li H. (2020). The Third Generation Anti-HER2 Chimeric Antigen Receptor Mouse T Cells Alone or Together With Anti-PD1 Antibody Inhibits the Growth of Mouse Breast Tumor Cells Expressing HER2 in vitro and in Immune Competent Mice. Frontiers in Oncology, 10, 1143.
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2

ADAMTS Immunohistochemistry Protocol

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All experimental steps were performed in accordance to the protocols recommended for the anti-human ADAMTS1, 8, 9, and 18 polyclonal antibodies (Abcam). After being deparaffinised at 65°C in a heat chamber and rehydrated, sections were subjected to epitope retrieval in 10× EDTA buffer (pH 8.0) at 110°C for 30 min. Subsequently, the sections were exposed to 3% H2O2 for 20 min to bleach endogenous peroxidases and were rinsed three times with phosphate-buffered saline (PBS) for 10 min. Sections were respectively incubated with a rabbit anti-human ADAMTSs (all 1 : 250 in BSA) for 1 h at 37°C, washed three times in PBS, and incubated in a biotinylated goat secondary anti-mouse polyclonal antibody for 15 min at 37°C. Following being washed in PBS, the tissues were visualised with 3,3′-diaminobenzidine tetrahydrochloride (DAB chromogen, Abcam) and counterstained with haematoxylin. Finally, the sections were dehydrated in graded ethanol, immersed in xylene, and coverslipped. All images were acquired using a 40× objective and an microscope (Leica).
Kılıç M.Ö., Aynekin B., Bozer M., Kara A., Haltaş H., İçen D, & Demircan K. (2017). Differentially regulated ADAMTS1, 8, 9, and 18 in pancreas adenocarcinoma. Przegla̜d Gastroenterologiczny, 12(4), 262-270.
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3

Immunohistochemistry and Immunofluorescence Protocols

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For both IHC and immunofluorescence (IF), 4-μm paraffin sections were deparaffinized with xylene. Slides were rehydrated using decreasing concentrations of ethanol. Antigen retrieval was performed at 121°C with the slides in either EDTA Ph9 or Citrate Ph6. Primary antibodies for GFPT2 (1:500; ph9; ab190966; Abcam), PU.1 (1:50; ph6; 554268; BD Biosciences), CD68 mouse (1:200; ph9; ab955; Abcam), CD68 rabbit (1:200; ph9; 76437S; Cell Signaling Technology), MMP9 (1:1,000; ph9; HPA001238; Millipore Sigma), YAP1 (1:1,200; ph6; sc-101199; Santa Cruz), desmin (1:40; D33 Dako), PDPN (D2–40 Dako) and Ki67 (1:50; ph9; Cell Signaling Technology) were used. For IHC immunodetection was completed using the Vectastain ABC kit (Vector Laboratories) and DAB chromogen (Abcam), according to the manufacturer's specifications. For IF a secondary mouse or rabbit Alexa488, Alexa555 or Alexa657 labeled antibodies were used (ThermoFisher). IHC and IF sections were scanned using the Keyence Fluorescence Microscope (BZ-X; Keyence) with the 40x lens, either using the fluorescent filter cube or the brightfield setting.
van IJzendoorn D.G., Matusiak M., Charville G.W., Spierenburg G., Varma S., Colburg D.R., van de Sande M.A., van Langevelde K., Mohler D.G., Ganjoo K.N., Bui N.Q., Avedian R.S., Bovée J.V., Steffner R., West R.B, & van de Rijn M. (2022). Interactions in CSF1-Driven Tenosynovial Giant Cell Tumors. Clinical Cancer Research, 28(22), 4934-4946.
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4

Immunohistochemical Analysis of Liver Fibrosis

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The mouse liver tissues were fixed with 4% paraformaldehyde and prepared into 5 μm sections. The sections were incubated with 3% hydrogen peroxide for 15 min and then continued to incubate with 5% BSA for approximately 20 min. Next, the sections were incubated with anti-α-SMA (#19245, 1:500, Santa Cruz Biotechnology, USA) and anti-Collagen I (#72026, 1:200, Santa Cruz Biotechnology) overnight at 4 °C. The sections were further incubated with the specific secondary antibodies at room temperature for 1 h. Subsequently, the sections were treated with DAB chromogen (Abcam, Cambridge, UK) and re-stained with hematoxylin. All data were assessed using microscope (DS-U3, Nikon, Tokyo, Japan) and ImageJ software (National Institutes of Health, Bethesda, MD). A minimum of 5 representative fields per biological replicate were taken into analysis.
Li Z., Dong J., Wang M., Yan J., Hu Y., Liu Y., Pan Y, & Li H. (2022). Resveratrol ameliorates liver fibrosis induced by nonpathogenic Staphylococcus in BALB/c mice through inhibiting its growth. Molecular Medicine, 28, 52.
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5

Immunochemical Analysis of ASO Distribution in Mouse Brain

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The distribution of ASO in mouse brain tissues was assessed by immunochemistry as described previously [66 (link)]. Briefly, fixed brain sections from 3 mice per group were digested for 1 min at 37 °C with a solution containing 500 mg/mL of proteinase K (Qiagen), 10 mM Tris-Cl, pH 7.5 (Wisent Bioproducts), 10 mM EDTA (Millipore Sigma), 2% SDS (Roche Diagnostics), 500 mM NaCl (Thermo Fisher Scientific), and 1.5 mM MgCl2 (Thermo Fisher Scientific). They were then blocked with 3% BSA for 30 min. After two washes in phosphate-buffered saline, the samples were incubated with a polyclonal rabbit anti-ASO primary antibody (6651 Pan ASO; Ionis Pharmaceuticals Inc., Carlsbad, CA, USA) for 1 h at room temperature and then with a goat anti-rabbit IgG (HRP) secondary antibody. The DAB chromogen (Abcam, Toronto, ON, Canada) was applied for 5 min before nuclear counterstaining with hematoxylin.
Ait Benichou S., Jauvin D., De Serres-Bérard T., Pierre M., Ling K.K., Bennett C.F., Rigo F., Gourdon G., Chahine M, & Puymirat J. (2022). Antisense oligonucleotides as a potential treatment for brain deficits observed in myotonic dystrophy type 1. Gene Therapy, 29(12), 698-709.
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6

Immunohistochemical Analysis of Chondrogenic Differentiation

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Antigen retrieval was performed in 10 mM sodium citrate buffer (pH 6.0). When staining for Collagen II and Collagen X, samples were incubated in 40mU/ml chondroitinase ABC (Sigma). Primary antibodies were diluted in 10% goat serum at concentrations detailed in Table 1. Staining was developed using DAB + Chromogen (Abcam).

Details of antibodies, concentrations and incubation times used to analyse protein deposition in chondrogenic MSC pellets by immunohistochemistry.

AntibodyManufacturerchondroitinase ABC TreatedAntibody ConcentrationIncubation
Collagen IIAbcam #54236YesPre-diluted4 °C 18 hrs
RUNX2Abcam #76956No1:10037 °C 1 hr
MEF2CAbcam #64644No1:2004 °C 18 hrs
Collagen XAbcam #49945Yes1:10004 °C 18 hrs
PTHR1Abcam# 104832No1:2004 °C 18 hrs
Browe D.C., Coleman C.M., Barry F.P, & Elliman S.J. (2019). Hypoxia Activates the PTHrP –MEF2C Pathway to Attenuate Hypertrophy in Mesenchymal Stem Cell Derived Cartilage. Scientific Reports, 9, 13274.
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