3 3 diaminobenzidine (dab)

Manufactured by Roche

Sourced in Germany, United States

DAB is a chromogenic substrate used in immunohistochemistry and in situ hybridization techniques to detect target proteins or nucleic acid sequences. It produces a brown precipitate at the site of the target, enabling visualization and analysis.

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Diaminobenzidine (DAB) (9 citations) 3,3′-diaminobenzidine (DAB) detection kit (8 citations) 3,3′-diaminobenzidine (DAB) substrate (4 citations) IVIEW 3,3-diaminobenzidine (DAB) detection kit (3 citations) 3,3'-diaminobenzidine tetrahydrochloride (DAB; (2 citations)

28 protocols using 3 3 diaminobenzidine (dab)

Immunohistochemical Analysis of Interneuron Markers

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For single CDH13 staining, 20 μm sections of perfusion-fixed brains on slides were incubated with primary antibody. N-Histofine Simple Stain Mouse MAX PO (G) (Nichirei Biosciences, Tokyo, Japan) and 3,3′-diaminobenzidine (Roche Diagnostics Deutschland, Mannheim, Germany) were used for detection.
For single staining of interneuronal markers, immersion-fixed brains (n=7–9 per genotype) were serially sectioned into 50 μm free-floating coronal sections. Sections were treated with heat-induced epitope retrieval, incubated with primary antibodies against SOM, PV and nNOS and detected using biotinylated secondary antibodies (Vector Laboratories, Burlingame, CA, USA), AB complex (Vector Laboratories) and 3,3′-diaminobenzidine (Roche Diagnostics Deutschland) development. Stained sections were then analyzed using Stereo Investigator v.11 (MBF Bioscience, Williston, VT, USA). PV-, SOM- and nNOS-positive cells in the stratum oriens (SO) were counted with the optical fractionator method.

Rivero O., Selten M.M., Sich S., Popp S., Bacmeister L., Amendola E., Negwer M., Schubert D., Proft F., Kiser D., Schmitt A.G., Gross C., Kolk S.M., Strekalova T., van den Hove D., Resink T.J., Nadif Kasri N, & Lesch K.P. (2015). Cadherin-13, a risk gene for ADHD and comorbid disorders, impacts GABAergic function in hippocampus and cognition. Translational Psychiatry, 5(10), e655-.

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Immunohistochemistry and TUNEL Assay for p21

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Immunohistochemistry (IHC) was performed according to standard protocols. Cultured palates were fixed in 4% paraformaldehyde for 24 h at room temperature, embedded in paraffin and sliced to generate 3-µm-thick serial sections. The sections were deparaffinized in xylene and rehydrated using a gradient of alcohol. Rabbit polyclonal antibodies were used as primary antibodies for p21 (ab109199; 1:250; Abcam, Cambridge, UK) staining. A two-step IHC detection reagent kit (SPN9001; ZSGB-BIO, Beijing, China) was used to localize the primary antibody. Subsequently, all the sections were treated with 3,3′-diaminobenzidine (ZSGB-BIO) under the microscope and stained with hematoxylin (ZSGB-BIO) for 30 sec. TUNEL assay was performed using a commercial kit (TUN11684817; Roche Diagnostics GmbH, Mannheim, Germany) according to standard procedure and the nuclei were stained with 3,3′-diaminobenzidine (ZSGB-BIO). All of the sections were viewed directly using an Axioskop 40 (Carl Zeiss AG, Oberkochen, Germany) microscope (magnification, ×10 and ×20).

Zhang Y.D., Dong S.Y, & Huang H.Z. (2017). Inhibition of periderm removal in all-trans retinoic acid-induced cleft palate in mice. Experimental and Therapeutic Medicine, 14(4), 3393-3398.

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Multiplex Immunohistochemistry for Mast Cells and Endothelial Cells

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Tryptase was used to detect MCs; CD34 staining, an endothelial cell marker, was used to visualize the glomerular and tubular interstitial compartments. The corresponding second section of each biopsy was stained by chromogenic multiplex staining: briefly, after de-paraffinization, CC1 (#950-124; Ventana Medical Systems, Tucson, AZ) antigen retrieval was performed for 64 min at 95°C, followed by incubation for 8 min with the Discovery inhibitor (#760-4840; Ventana Medical Systems). Primary antibody CD34 (#790-2927; Ventana Medical Systems) was incubated for 32 min at 37°C, followed by detection with OmniMap goat-anti-rabbit HRP (#760-4311; Ventana Medical Systems), and visualized using purple kit for 32 min. A CC2 (#950-123, Ventana Medical Systems) 100°C stripping step was performed for 8 min. Tryptase (#760-4276; Ventana Medical Systems) was incubated at 37°C for 32 min, followed by secondary antibody, OmniMap goat-anti-mouse HRP (#760-4310; Ventana Medical Systems) at 37°C for 24 min, and visualized with 3,3′-Diaminobenzidine (#760-229; Ventana Medical Systems) for 32 min. Finally, Hematoxylin II (#790-2208; Ventana Medical Systems) was used to counter stain for 8 min and then a blue coloring reagent (#760-2037; Ventana Medical Systems) for 4 min according to the manufactures instructions (Ventana Medical Systems).

Varol H., van der Elst G., Baan C.C., van Baardwijk M., Hesselink D.A., Duong van Huyen J.P., Kramann R., Rabant M., van den Bosch T.P, & Clahsen-van Groningen M.C. (2023). Mast Cells in Kidney Transplant Biopsies With Borderline T Cell-mediated Rejection and Their Relation to Chronicity. Transplantation Direct, 9(5), e1480.

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Breast Tumor Specimen Preparation and Analysis

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Deidentified breast tumor specimens exhibiting multiple morphological features were selected from archival FFPE tissue blocks at the Biospecimen Resource Shared Services at Rutgers Cancer Institute of New Jersey (IRB Protocol 009601). After a board-certified pathologist evaluated the quality of the specimens, tissue blocks were sectioned serially at 4 μm thickness. Two sections (#1 and #2) were immunohistochemically stained with estrogen receptor and progesterone receptor antibody (not shown). Section #3 was double stained with transformation-related protein (P63) in brown [3,3′-diaminobenzidine (DAB)] and α-smooth muscle actin (SMA) in red (RedMap; Ventana Medical Systems). Section #5 was de-paraffinized, but not stained, and kept in phosphate-buffered saline at room temperature before AFM probing. Section #6 was stained with hematoxylin and eosin (H&E) and once again quality controlled by a pathologist.

Chen W., Brandes Z., Roy R., Chekmareva M., Pandya H.J., Desai J.P, & Foran D.J. (2015). Robot-Guided Atomic Force Microscopy for Mechano-Visual Phenotyping of Cancer Specimens. Microscopy and microanalysis : the official journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada, 21(5), 1224-1235.

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Immunohistochemical Assay for Annexin A1

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Briefly, 4 μm paraffin mouse lung tissue sections were mounted on glass slides and were deparaffinized. The immunostaining was performed on the Ventana Discover ULTRA autostainer (Ventana Medical Systems, Oro Valley, AZ) using OmniMapHRP detection method. The primary antibody was anxA1 antibody (clone 4-huIgG1) and was incubated with concentration at 0.5 μg/mL Following primary antibody incubation, the samples were incubated in the specific link antibody rabbit anti-human IgG at concentration 2 μg/ml (Jackson ImmunoResearch Laboratories® cat# 309-005-082) for 16 minutes. Then, primary antibody was visualized with OmniMap goat anti-rabbit HRP (catalog no. Cat #760–4311, Ventana) respectively, and DAB (catalog no. cat# 760–159, Ventana).
All IHC stained slides were assessed by an experienced board-certified pathologist (JAC). The intensity of Annexin A1 IHC staining was assessed semi-quantitatively: 0 (none), 1+ (faint) 2+ (moderate), 3+ (maximum); and the extent of staining was estimated as the percentage of tissue stained positively. In the tumor samples, neoplastic cell staining was assessed as present or absent.

Allen K.L., Cann J., Zhao W., Peterson N., Lazzaro M., Zhong H., Wu H., Dall’Acqua W.F., Borrok M.J., Damschroder M.M., Tsui P, & Li Q. (2020). Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non–small-cell lung carcinoma and rodent tumor models. PLoS ONE, 15(6), e0234268.

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Immunohistochemical Staining Protocol

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Slides were immunostained according to the manufacturer’s recommendations and in brief processed as follows: the slides were dried at 60°C for 1 hour and placed in the BenchMark Ultra instrument (Ventana), deparaffinized on-board and submitted to heat-induced epitope retrieval in cell conditioning 1 for 32 minutes at 95°C. Following endogenous peroxidase blocking, the primary antibody (rabbit monoclonal clone 4B5, 760-2991) was applied for 20 minutes at 36°C. After a wash in buffer the visualization complex, UltraView DAB (horseradish peroxidase -labeled multimer, Ventana, 760-500) was applied, and after a new wash in the buffer, the slides were finally developed with DAB (Ventana, 760-500) and counterstained with hematoxylin II (Ventana, 790-2208).

Nielsen S.L., Nielsen S, & Vyberg M. (2015). Digital Image Analysis of HER2 Immunostained Gastric and Gastroesophageal Junction Adenocarcinomas. Applied Immunohistochemistry & Molecular Morphology, 25(5), 320-328.

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Immunocytochemical Staining of VCAN

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The cultured cells were washed with PBS and cytospun on slides for 15 min, then fixed with 4% paraformaldehyde for 20 min and permeabilized with 1% Triton for 20 min at room temperature. Following blocking with 1% bovine serum albumin in PBS for 30 min, cells were immunostained with rabbit monoclonal VCAN antibody (Abcam, USA, 1:200, Cat No: ab177480) overnight at 4 °C. The primary antibody was revealed using the Ventana Optiview DAB (3,3'-Diaminobenzidine) Detection System. The slides were subsequently counterstained with Mayer's hematoxylin and analyzed using a bright field microscope.

Yang L., Wang L., Yang Z., Jin H., Zou Q., Zhan Q., Tang Y., Tao Y., Lei L., Jing Y., Jiang X, & Zhang L. (2019). Up-regulation of EMT-related gene VCAN by NPM1 mutant-driven TGF-β/cPML signalling promotes leukemia cell invasion. Journal of Cancer, 10(26), 6570-6583.

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Immunohistochemical Analysis of Annexin A1

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Briefly, 4 μm paraffin mouse lung tissue sections were mounted on glass slides and were deparaffinized. The immunostaining was performed on the Ventana Discover ULTRA autostainer (Ventana Medical Systems, Oro Valley, AZ) using OmniMapHRP detection method. The primary antibody was anxA1 antibody (clone 4-huIgG1) and was incubated with concentration at 0.5 μg/mL Following primary antibody incubation, the samples were incubated in the specific link antibody rabbit anti-human IgG at concentration 2 μg/ml (Jackson ImmunoResearch Laboratories1 cat# 309-005-082) for 16 minutes. Then, primary antibody was visualized with OmniMap goat anti-rabbit HRP (catalog no. Cat #760-4311, Ventana) respectively, and DAB (catalog no. cat# 760-159, Ventana).
All IHC stained slides were assessed by an experienced board-certified pathologist (JAC). The intensity of Annexin A1 IHC staining was assessed semi-quantitatively: 0 (none), 1+ (faint) 2+ (moderate), 3+ (maximum); and the extent of staining was estimated as the percentage of tissue stained positively. In the tumor samples, neoplastic cell staining was assessed as present or absent.

Allen K., Cann J., Zhao W., Peterson N., Lazzaro M., Zhong H., Wu H., Dall’Acqua W.F., Borrok M.J., Damschroder M., Tsui P., & Li Q. (2020). Upregulation of annexin A1 protein expression in the intratumoral vasculature of human non–small-cell lung carcinoma and rodent tumor models. PLoS ONE, 15(6), e0234268-e0234268.

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Immunohistochemical Analysis of 11β-HSD1 and CYP11B1 in Lung Cancer

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Investigations on human patient samples have been conducted in accordance with the local ethics committees (ethics committees of the Canton Thurgau and Eastern Switzerland) and the Declaration of Helsinki and informed consent was obtained from all subjects. Formalin-fixed and paraffin-embedded lung cancer tissue sections were provided by Achim Fleischmann (Kantonspital Münsterlingen, Switzerland). Heat-induced antigen retrieval was performed in sodium citrate pH 6,0 (anti-CYP11B1 antibody) or Tris-EDTA pH 9,0 (anti-11β-HSD1 antibody) buffer. Endogenous peroxidase was blocked with 1% hydrogen peroxide (Sigma-Aldrich). The sections were stained using a rabbit anti-human 11β-HSD1 antibody (ab39364, Abcam), rabbit anti-human CYP11B1 antibody (HPA056348, Sigma) or rabbit isotype control, and a biotin-labelled secondary antibody. Vectastain ABC-kit (Vector Laboratories, Burlingame, CA, US) was used to convert the substrate DAB (Roche) into a brown colored product. Nuclei were visualized with hematoxylin (Roth).

Merk V.M., Grob L., Fleischmann A, & Brunner T. (2023). Human lung carcinomas synthesize immunoregulatory glucocorticoids. Genes and Immunity, 24(1), 52-56.

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Immunohistochemical Analysis of UPR Markers

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Immunohistochemical analysis was performed for assessing the expression of GRP78, CHOP and Caspase‐12. In brief, after deparaffinization, endogenous peroxidases in the sections were blocked with 0.3% hydrogen PBS, followed by treatment with 1% bovine serum albumin for 60 min. at room temperature. Goat monoclonal anti‐GRP78, anti‐CHOP or anti‐Caspase‐12 primary antibodies at 1:50 dilution were added to the sections overnight at 4°C. The sections were washed with PBS and incubated with biotinylated secondary antibody and streptavidin‐horseradish peroxidase solution. After washing with PBS, sections were incubated with DAB (Roche, Mannheim, Germany) for 5 min., and counterstained with haematoxylin. PBS instead of primary antibody was used to serve as the negative control. The stained sections were visualized using a light microscope (Olympus BX51; Olympus Co.) at 400× and analysed with a computer‐assisted colour image analysis system (Image‐ProPlus 6.0; Media Cybernetics). The density of immunopositive nuclei was determined randomly in five microscopic fields per section and the average optical density was calculated for each group.

Yu H., Zhen J., Yang Y., Gu J., Wu S, & Liu Q. (2016). Ginsenoside Rg1 ameliorates diabetic cardiomyopathy by inhibiting endoplasmic reticulum stress‐induced apoptosis in a streptozotocin‐induced diabetes rat model. Journal of Cellular and Molecular Medicine, 20(4), 623-631.

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