Dab substrate

Manufactured by Roche

Sourced in Germany, Switzerland

DAB substrate is a chromogenic detection reagent commonly used in immunohistochemistry and immunocytochemistry applications. It produces a brown colored precipitate upon reaction with the enzyme label, allowing for the visualization of target antigens in biological samples.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (2 mentions) Rabbit anti mouse igg hrp conjugate, by Merck Group (1 mentions) Vectastain abc kit, by Vector Laboratories (1 mentions) In situ cell death detection kit, by Roche (1 mentions) Hier buffer h, by Thermo Fisher Scientific (1 mentions) Colorimetric assay kit, by Abcam (1 mentions) Apo one homogeneous caspase 3 7 assay, by Promega (1 mentions) Nanozoomer sq digital slide scanner, by Hamamatsu Photonics (1 mentions) Immpress hrp horse anti rabbit igg polymer detection kit, by Vector Laboratories (1 mentions) Rq1 dnase, by Promega (1 mentions) Anti actin hrp antibody, by Santa Cruz Biotechnology (1 mentions) Ripa buffer, by Roche (1 mentions) Anti hoxd10, by Santa Cruz Biotechnology (1 mentions) Anti pten, by Santa Cruz Biotechnology (1 mentions) Anti bim, by Cell Signaling Technology (1 mentions) Dnase, by Roche (1 mentions) Protease inhibitor cocktail, by Roche (1 mentions) Primary monoclonal antibody, by Santa Cruz Biotechnology (1 mentions) Fluorescein isothiocyanate (fitc), by Santa Cruz Biotechnology (1 mentions) Mouse monoclonal antibodies, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

DAB peroxidase substrate 3,3′-diaminobenzidine (DAB) substrate DAB substrate kit Liquid DAB substrate kit

15 protocols using dab substrate

Western Blot Analysis of GFLV Movement Protein

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Infected and non-infected leaf tissues were ground in 1 × PBS, mixed with an equal volume of 2 × sample buffer and boiled for 5 min. Next, the denatured crude leaf extracts, purified GFLV MP and the protein from non-induced bacteria were separated by 12% SDS-PAGE, blotted onto nitrocellulose membranes using electro-blotter at 80 V for 120 min at room temperature as mentioned previously. After blocking with 5% powdered skim milk in phosphate buffered saline Tween 20 (PBST), the membrane was incubated with the anti-GFLV MP IgG (1:500) overnight at 4°C with gentle shaking. After 4 × washing with PBST, the membrane was incubated with the secondary antibody, mouse anti-rabbit IgG HRP-conjugate (1:1,000; Sigma-Aldrich, St. Louis, MO, USA) at room temperature for about 2 h. The bands of interest were visualized by reaction with freshly prepared DAB substrate (Roche Applied Science, Indianapolis, IN, USA).
This study was approved by the animal ethics committee of Pasteur Institute of Iran (IPI) (approval No. 66465132).

Koolivand D., Bashir N.S., Behjatnia S.A, & Joozani R.J. (2016). Production of Polyclonal Antibody against Grapevine fanleaf virus Movement Protein Expressed in Escherichia coli. The Plant Pathology Journal, 32(5), 452-459.

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Nonfluorescent In Situ Hybridization Protocol

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Nonfluorescent ISHs were carried out at 37° using 100−150 ng of biotin-labeled probe on each chromosomal slide, following the protocol of Biémont et al. (2004) (link) with minor modifications: 1) The slides were washed in 70% ethanol before the later washing with 95% ethanol in the pre-hybridization step; 2) two washes with 2× saline sodium citrate at 37° for 10 min and one wash for a few seconds in 2× saline sodium citrate at room temperature after the hybridization step; 3) one wash with 1× phosphate-buffered saline (PBS) just after the washes with 0.1% Triton in 1× PBS after the detection step; 4) one wash with 0.1% Triton in 1× PBS before the wash with 1× PBS after the revelation step.
Hybridization signals were detected and revealed using a Vectastain ABC KIT (Vector Laboratories) and DAB Substrate (Roche), respectively. Then, the slides were stained with Giemsa solution (5%) for 5 min and made permanent with EUKITT (Panreac). Finally, the positives hybridization signals were mapped in the polytene chromosome of the sequenced strain Gd-H4-1 and recorded with a Moticam Package (Moticam3 with 3.0 MP and Motic Images Plus 2.0). Their physical locations were determined according to current photomap of the species (Schaeffer et al. 2008 (link)) and the chromosomal analysis of this strain by Rohde and Valente (2012) (link).

Garcia C., Delprat A., Ruiz A, & Valente V.L. (2015). Reassignment of Drosophila willistoni Genome Scaffolds to Chromosome II Arms. G3: Genes|Genomes|Genetics, 5(12), 2559-2566.

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Apoptosis Assay of Embryos

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MO-injected embryos and uninjected controls were assayed for apoptosis by TUNEL using the In Situ Cell Death Detection Kit (Roche, Mannheim, Germany) for whole-mount embryos. The embryos were fixed with 4% PFA and treated with Proteinase K (10 μg/ml) for 5 minutes prior to the assay. Then the fluorescent signal was transformed to a light signal with DAB Substrate (Roche), and the embryos were cleared in PBS/glycerol (1:1) for viewing with a light microscope.

Ma L., Parkhurst A, & Jeffery W.R. (2014). The role of a lens survival pathway including sox2 and αA-crystallin in the evolution of cavefish eye degeneration. EvoDevo, 5, 28.

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Immunohistochemical Staining Protocol

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The tissue sections were dehydrated through graded alcohols and embedded in paraffin wax. The slides were heated in Dewax and HIER Buffer H (Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 20 min, and endogenous peroxidase activity with 3% H₂O₂ was blocked, followed by blocking with 5% BSA for 10 min. The sections were then incubated with primary antibody overnight at 4 °C. The sections were treated with a biotinylated secondary antibody for 1 h at 37 °C. DAB Substrate (Hoffmann-La Roche Ltd., Basel, Switzerland) was then applied on the sections for 30 min. Finally, sections were washed with PBS and then incubated in hematoxylin for 15 min.

Huang P.C., Kuo W.W., Shen C.Y., Chen Y.F., Lin Y.M., Ho T.J., Padma V.V., Lo J.F., Huang C.Y, & Huang C.Y. (2016). Anthocyanin Attenuates Doxorubicin-Induced Cardiomyotoxicity via Estrogen Receptor-α/β and Stabilizes HSF1 to Inhibit the IGF-IIR Apoptotic Pathway. International Journal of Molecular Sciences, 17(9), 1588.

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Immunohistochemical Staining Protocol

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For immunohistochemical staining, the slides were kept in boiling citrate buffer (pH 6.0) for 30 min and were then blocked with serum and then incubated with primary antibody overnight at 4 °C. Biotinylated secondary antibody was applied on the sections and left for 1 h at room temperature. The sections were treated with DAB Substrate (Hoffmann-La Roche Ltd, Basel, Switzerland) for 5 to 15 min, and photographs were taken using an Olympus P74 (Olympus, Waltham, MA, USA) microscope for analysis.

Marthandam Asokan S., Wang T., Su W.T, & Lin W.T. (2019). Antidiabetic Effects of a Short Peptide of Potato Protein Hydrolysate in STZ-Induced Diabetic Mice. Nutrients, 11(4), 779.

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Immunohistochemical Staining of Skin Tissue

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The embedded 0.5 μm-thick skin tissue samples was dried at 60°C overnight following dewaxing in xylene for 30 min, and rehydration in 100% ethanol for 30 min. Endogenous peroxidase activity was blocked by incubation in hydrogen peroxide blocking buffer (3% H₂O₂) for 10 min. After rinsing the sections with tap water for 15 min, nonspecific binding was blocked by incubation with 2.5% horse serum for 10 min. Then, the sections were incubated with primary antibodies (1:100 dilution) overnight at 4 °C. After washing with 1× PBS, the samples were treated with secondary antibody for 10 min at RT. Subsequently, HRP Polymer was added to the slides for 10 min at RT, followed by DAB substrate (Roche) application for 15 s at RT. Then, the sample was stained with hematoxylin for 5 min at RT. Finally, the tissue–polymer complex was detected using a light microscope (Olympus).

Lee J.J., Ng S.C., Ni Y.T., Liu J.S., Chen C.J., Padma V.V., Huang C.Y, & Kuo W.W. (2021). Protective effects of galangin against H2O2/UVB-induced dermal fibroblast collagen degradation via hsa-microRNA-4535-mediated TGFβ/Smad signaling. Aging (Albany NY), 13(23), 25342-25364.

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Western Blotting of GFP Proteins

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For western blotting, the cells were harvested by trypsin, and the cell pellets were resuspended in PBS. Total cellular proteins were solved in 6X sample buffer containing Tris-HCl (0.5 M), glycerol, SDS, and 2-Mercaptoethanol (2%). The samples were separated on 15% acrylamide gel and transferred to nitrocellulose membrane. The membrane was incubated in blocking buffer (TBS 10X, 0.1% Tween 20, BSA, Merck) and washed with TBS10X and 0.1% Tween 20. Then, anti-GFP polyclonal antibody conjugated with horseradish peroxidase (1:10000 v/v) was used to detect the proteins of interest in the presence of DAB substrate (Ro-che Diagnostics-Germany).

Basirnejad M., Bolhassani A, & Sadat S.M. (2018). The Distinct Role of Small Heat Shock Protein 20 on HCV NS3 Expression in HEK-293T Cell Line. Avicenna Journal of Medical Biotechnology, 10(3), 152-157.

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Liver Tissue Analysis of Triglycerides, Fatty Acids, Cholesterol, and Apoptosis

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Liver samples obtained at week 24 were employed. 100 mg of liver tissue was placed in 1 ml of PBS and homogenized using a Polytron Model PT 10–35 GT-D homogenizer (Kinematica, Luzern, Switzerland). The homogenate was then centrifuged at 10,000 x g for 5 minutes at 4 C and the supernatant retained for the assays. Liver triglycerides, free fatty acids, total and free cholesterol were measured using colorimetric assay kits per the manufacturer’s protocol (Biovision, Milpitas, CA). Caspase 3/7 activity was measured using the Apo-ONE Homogeneous Caspase-3/7 assay per the manufacture’s protocol (Promega, Madison, WI). Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed on a paraffin embedded sections using the fluorescein In Situ Cell Death Detection Kit, POD combined with DAB substrate per the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). Hepatocyte apoptosis was quantified by counting the number of TUNEL positive cells in twenty representative 200x microscopic fields across the section and the total number of cells per field was determined by counting the number of nuclei present in a representative quadrant in each field and multiplying by four. The apoptotic index was calculated as the number of TUNEL positive cells per 1000 cells.

Liang T., Alloosh M., Bell L.N., Fullenkamp A., Saxena R., Van Alstine W., Bybee P., Werling K., Sturek M., Chalasani N, & Masuoka H.C. (2015). Liver Injury and Fibrosis Induced by Dietary Challenge in the Ossabaw Miniature Swine. PLoS ONE, 10(5), e0124173.

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Quantifying Hepatocyte Apoptosis via TUNEL

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TdT-mediated dUTP-X nick end labeling (TUNEL) assay was performed on a paraffin-embedded sections using the Fluorescein In Situ Cell Death Detection Kit, POD combined with DAB substrate per the manufacturer’s protocol (Roche Applied Science, Indianapolis, IN). Hepatocyte apoptosis in liver sections was determined by counting the number of TUNEL positive cells in twenty representative 40x microscopic fields across the section and the total number of cells per field was determined by counting the number of nuclei present in a representative quadrant and multiplying by four. The apoptotic index was calculated as the number of TUNEL positive cells per 400 cells.

Masuoka H.C., Vuppalanchi R., Deppe R., Bybee P., Comerford M., Liangpunsakul S., Ghabril M, & Chalasani N. (2015). Individuals with Primary Sclerosing Cholangitis have Elevated Levels of Biomarkers for Apoptosis but Not Necrosis. Digestive diseases and sciences, 60(12), 3642-3646.

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Histological Scoring of Lung Inflammation

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Histological scoring was conducted as in [45 (link)], and expressed as the sum of the parameter described in S1 Table. Scoring was performed blindly by a pathologist. For IHC tissue sections were deparaffinized, rehydrated, and heated in citrate buffer (40mM sodium citrate dihydrate, 60mM citric acid, pH 6) and blocked with 1:50 Fc block reagent (rat anti-mouse CD16/CD32, IGC antibody facility, clone 2.4G2). Slides were then incubated with rabbit α-NP (102) 1:1000 for 16h at 4°C. After blocking of endogenous peroxidases sections were incubated with ImmPRESS HRP Horse Anti-Rabbit IgG Polymer Detection Kit (Vector Laboratories, MP-7401-15) for 1h at RT and then with DAB substrate (Roche, 11718096001) according to manufacturer’s instructions. Finally, lung sections were contrasted with Mayer Hematoxylin and images taken in a NanoZoomer-SQ Digital slide scanner (Hamamatsu Photonics).
Cell infiltrates in lung parenchyma were assessed semi-quantitatively by adding scores obtained for alveolar inflammation, interstitial inflammation and perivascular/peribronchiolar inflammation.

Santos N.B., Vaz da Silva Z.E., Gomes C., Reis C.A, & Amorim M.J. (2021). Complement Decay-Accelerating Factor is a modulator of influenza A virus lung immunopathology. PLoS Pathogens, 17(7), e1009381.

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