Dab peroxidase kit

Manufactured by Vector Laboratories

Sourced in United States, United Kingdom

The DAB peroxidase kit is a laboratory reagent used for the detection and visualization of peroxidase enzyme activity in various biological samples. It provides a 3,3'-diaminobenzidine (DAB) chromogenic substrate that produces a brown reaction product when catalyzed by the peroxidase enzyme. This kit is commonly used in immunohistochemistry, in situ hybridization, and other applications where the localization of peroxidase-labeled targets is required.

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Lab products found in correlation

Cas block, by Thermo Fisher Scientific (3 mentions) Streptavidin hrp, by BioLegend (2 mentions) Biotin anti tra 1 60, by Thermo Fisher Scientific (2 mentions) Avidin biotin complex elite, by Vector Laboratories (2 mentions) Cellsens software, by Olympus (2 mentions) Dp73 camera, by Olympus (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Anti p16 ink4a, by Thermo Fisher Scientific (1 mentions) Nanozoomer 2.0 rs slide scanner, by Hamamatsu Photonics (1 mentions) Hematoxylin h, by Vector Laboratories (1 mentions) Vectastain elite abc hrp kit, by Vector Laboratories (1 mentions) Cleaved caspase 3, by Cell Signaling Technology (1 mentions) Dm6000b microscope, by Leica (1 mentions) Sc 67261, by Santa Cruz Biotechnology (1 mentions) T per, by Thermo Fisher Scientific (1 mentions) Gapdh, by Cell Signaling Technology (1 mentions) Image lab, by Bio-Rad (1 mentions) Versadoc imaging system, by Bio-Rad (1 mentions) Hematoxylin, by Merck Group (1 mentions) Sc 9966, by Santa Cruz Biotechnology (1 mentions)

Close spelling variants of this product

ImmPRESS™ peroxidase secondary and DAB substrate kits Vectastain ABC and DAB peroxidase substrate kits 3,3′-diaminobenzidine (DAB) peroxidase substrate kit DAB substrate kit for peroxidase visualization of secondary antibodies DAB (3-3′ diaminobenzidine) peroxidase kit DAB Peroxidase Substrate detection kit Diaminobenzidine (DAB) peroxidase substrate kit DAB Peroxidase Substrate Kit ImmPACT DAB Peroxidase Substrate Kit Vector DAB Peroxidase Substrate Kit

17 protocols using dab peroxidase kit

Immunohistochemical Analysis of Cellular Markers

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Sections were deparaffinized and rehydrated using three changes of xylenes followed by ethanol gradient. Antigen retrieval was performed using citrate solution pH 6.0 at 60 ℃ overnight, followed by blocking for 1 h using blocking solution (10% goat serum in PBS, 1% tween-20, 1% BSA). Sections were then incubated with primary antibody diluted in DAKO dilution solution (S3022, Agilent, Santa Clara, CA) overnight at 4 degrees then washed and incubated with biotin-conjugated secondary antibody (BP-1100, Vector Biolabs, Burlingham, CA) at room temperature for 2 h. Sections were washed and incubated with streptavidin-HRP using Vectastain ABC-HRP (PK-4000, Vector Laboratories, Burlington, CA) for 20 min. After washing, sections were developed using DAB peroxidase kit (SK4100, Vector Laboratories, Burlingham, CA). Anti-p16-ink4a (PA5-20379, Thermo Fisher), anti-IκB-ζ (NBP-89835, Novus Biologicals, Centennial, CO), anti-3-NT (06-284, Millipore Sigma, Burlington, MA), cleaved caspase-3 (9661 S, CST, Danvers, MA), and Υ-H2AX (2577 S, CST, Danvers, MA) were used in blocking solution (1:100 dilution).

Arra M., Swarnkar G., Alippe Y., Mbalaviele G, & Abu-Amer Y. (2022). IκB-ζ signaling promotes chondrocyte inflammatory phenotype, senescence, and erosive joint pathology. Bone Research, 10, 12.

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Immunohistochemical Analysis of Lung Tissue

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Hematoxylin and eosin (H&E) staining and immunohistochemistry were performed on paraffin-embedded lungs using standard protocols. Immunohistochemistry was performed using antibodies directed against Ki67 (1:100, BD Biosciences,) and Cleaved caspase 3 (1:400, Cell Signaling Technologies). Briefly, paraffin sections were re-hydrated, unmasked in 10mM Sodium Citrate buffer with 0.05% Tween 20 in a pressure cooker for 10 minutes, the peroxidase was quenched for 15 minutes in 3% H₂O₂, sections were blocked for 30 minutes in TBS with 0.025% Triton X-100 supplemented with 10% serum and 1% BSA, and incubated overnight at 4°C with primary antibody. On the next day, the sections were incubated for 30 minutes with biotinylated antibody compatible with the primary antibody used (1:1000, Vector Laboratories) and were subsequently incubated with VECTASTAIN Elite ABC HRP Kit (Vector Laboratories), according to manufacturer’s instructions. The sections were washed with TBS in between steps. Staining was performed using the DAB peroxidase kit (Vector Laboratories) and hematoxylin (H-3401, Vector Laboratories) for counter-staining. A Leica DM6000B microscope (Leica Microsystems) or NanoZoomer 2.0-RS slide scanner (Hamamatsu) was used for imaging.

Bieging-Rolett K.T., Kaiser A., Morgens D.W., Boutelle A.M., Seoane J.A., Van Nostrand E.L., Zhu C., Houlihan S.L., Mello S.S., Yee B.A., McClendon J., Pierce S.E., Winters I.P., Wang M., Connolly A.J., Lowe S.W., Curtis C., Yeo G.W., Winslow M.M., Bassik M.C, & Attardi L.D. (2020). Zmat3 is a key splicing regulator in the p53 tumor suppression program. Molecular cell, 80(3), 452-469.e9.

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Reprogramming Fibroblasts to iPSCs

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Reprogramming assays were performed essentially as described previously⁹. Briefly, dH1f fibroblasts were seeded at 2.5 × 10⁵ cells/well in a 12-well plate and transduced overnight with a pool of lentiviral (OCT4, SOX2, NANOG) and retroviral (LIN28A variants) particles. Six days later, cells were trypsinized and 1–2 × 10⁵ cells/well were re-plated onto MEF-coated 12-well plates. Medium was switched to hESC medium and changed daily until day 21 when reprogramming efficiency was measured. To do so, cells were fixed with 4% paraformaldehyde and stained with biotin-anti-TRA-1-60 (eBioscience #13-8863-82; 1:250) and streptavidin-HRP (Biolegend #405210; 1:500) primary and secondary antibodies, respectively. Staining was developed with the DAB Peroxidase kit (Vector Labs), and the number of iPSC colonies was quantified using ImageJ. Experiments were carried out and analyzed in a blinded manner.

Tsanov K.M., Pearson D.S., Wu Z., Han A., Triboulet R., Seligson M.T., Powers J.T., Osborne J.K., Kane S., Gygi S.P., Gregory R.I, & Daley G.Q. (2016). LIN28 phosphorylation by MAPK/ERK couples signaling to the post-transcriptional control of pluripotency. Nature cell biology, 19(1), 60-67.

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Western Blot and Immunohistochemistry of CYP27B1

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For western blot, frozen tissues were homogenized in T-Per (Thermo Fisher) and quantitated with a bicinchoninic acid (BCA) protein assay kit (Thermo Fisher). Sixty micrograms of protein were electrophoresed in an SDS-polyacrylamide gel, blotted and probed with antibodies to the following targets: CYP27B1 (sc-67261, Santa Cruz Biotechnology) and GAPDH (Cell Signaling). Densitometry was done with the VersaDoc Imaging System (Bio-Rad) and quantified using Image Lab (Bio-Rad). Densitometry values for CYP27B1 were normalized to those of the housekeeping control (GAPDH) for each lane. Three normal controls were run on each blot and CYP27B1 values for disease samples were expressed as a fold-difference from the mean value of the normal samples. For immunohistochemistry, 10 micron paraffin sections were used. Deparaffinized sections were immersed in 10 mM citrate buffer (pH 6.0) heated at 100°C for 30 min, and allowed to cool to room temperature. After antigen retrieval, all sections were treated with 3% H2O2 for 10min. After blocking, sections were incubated with CYP27B1 antibodies (ab95047, Abcam) overnight at 4°C. Slides were incubated for an hour at room temperature with OneStep polymer HRP anti-rabbit secondary antibody (GTX83399, Genetex, Irvine). After rinsing with PBS, slides were developed with a DAB peroxidase kit (Vector Laboratories) and counterstained with hematoxylin.

Si Y., Kazamel M., Kwon Y., Lee I., Anderson T., Zhou S., Bamman M., Wiggins D., Kwan T, & King P.H. (2020). The Vitamin D Activator CYP27B1 is Upregulated in Muscle Fibers in Denervating Disease and can Track Progression in Amyotrophic Lateral Sclerosis. The Journal of steroid biochemistry and molecular biology, 200, 105650.

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Histological and Immunohistochemical Analysis

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Tissues were fixed with 4% formalin, embedded in paraffin embedding and sectioning then stained with hematoxylin and eosin for histological. For immunohistochemistry (IHC), paraffin-embedded tissues were deparaffinized, followed by antigen retrieval using 25 mM citrate buffer (pH 6) in a pressure cooker. Then The sections were left to cool for 25 min, followed by blocking of the endogenous peroxidase with 3% H₂O₂ for 15 min. To reduce nonspecific binding of the primary antibody, tissues were blocked with blocking solution (CAS Block) (Invitrogen, San Diego, CA), followed by incubation with the primary antibody overnight at 4 °C. Sections were washed with TBST and incubated with secondary anti-rabbit, anti-mouse, or anti-rat immunoglobulin antibody for 30 min. The reaction was then performed using a DAB peroxidase kit (Vector Laboratories, SK-4100, Mowry Ave Newark, United State), followed by hematoxylin stain for 45 s as a counterstain. IHC stains were manually counted, at least five pictures were taken for each slide, and the average was calculated for each slide.

Husanie H., Abu-Remaileh M., Maroun K., Abu-Tair L., Safadi H., Atlan K., Golan T, & Aqeilan R.I. (2022). Loss of tumor suppressor WWOX accelerates pancreatic cancer development through promotion of TGFβ/BMP2 signaling. Cell Death & Disease, 13(12), 1074.

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Immunohistochemical Analysis of Mouse Tissues

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Tissues collected from different mice at different ages were fixed in 4% formalin. Paraffin‐embedded tissue sections were deparaffinized and rehydrated. Antigen retrieval was performed in 25 mM sodium citrate buffer pH 6.0 (for WWOX, GFAP, Iba1) using pressurized chamber for 2.5 min. Endogenous peroxidase was blocked with 3% H₂O₂ for 15 min. The sections were then incubated with blocking solution (CAS Block) for 30 min to reduce non‐specific binding followed by incubation with the primary antibody. Slides were subsequently incubated with horseradish peroxidase‐conjugated anti‐rabbit or anti‐mouse immunoglobulin antibody for 30 min. The enzymatic reaction was detected in a freshly prepared 3,3 diamminobenzidine using DAB peroxidase kit (Vector laboratories) for several min at room temperature. The sections were then counterstained with hematoxylin.

Repudi S., Kustanovich I., Abu‐Swai S., Stern S, & Aqeilan R.I. (2021). Neonatal neuronal WWOX gene therapy rescues Wwox null phenotypes. EMBO Molecular Medicine, 13(12), e14599.

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Reprogramming Fibroblasts to iPSCs

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Partial Decalcification and IHC Staining

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Fixed bones and tumors [4% formalin] were partially decalcified in 14% EDTA for 3 days, followed by paraffin embedding, sectioning then staining with hematoxylin (Sigma)) and eosin (Sigma) (H&E). For Immunohistochemistry, paraffin-embedded tissues were deparaffinized, followed by antigen retrieval with 25 mM citrate buffer (PH 6) in a pressure cooker. Then, the sections were left to cool for 25 min, followed by blocking of the endogenous peroxidase with 3% H₂O₂ (Sigma) for 15 min. To reduce non-specific binding of the primary antibody, tissues were blocked with blocking solution (CAS Block) (Invitrogen), followed by incubation with the primary antibody overnight at 4 °C. Sections were washed with TBST, followed by incubation with secondary anti-mouse immunoglobulin antibody for 30 min (Nichirei Biosciences). The reaction was then performed using a DAB peroxidase kit (Vector Laboratories, SK-4100), followed by hematoxylin (Bar Naor, Cat# BN4537N) staining for 40 s as a counterstain. IHC stains were manually counted, at least five pictures were taken for each slide, and the average was calculated for each slide. The antibodies used were mouse monoclonal MCM7 (141.2) [sc-9966-Santa Cruz 1:200].

Akkawi R., Hidmi O., Haj-Yahia A., Monin J., Diment J., Drier Y., Stein G.S, & Aqeilan R.I. (2024). WWOX promotes osteosarcoma development via upregulation of Myc. Cell Death & Disease, 15(1), 13.

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Immunohistochemical Analysis of Tissue Samples

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Tissues were fixed with 4% formalin, then 70% ethanol and processed. Paraffin-embedded tissue sections were deparaffinized and rehydrated, then stained with H&E for histological observation and diagnosis. For IHC, paraffin-embedded tissues were deparaffinized, followed by antigen retrieval using 25 mM citrate buffer (pH 6) in a pressure cooker. Then The sections were left to cool for 25 min, followed by blocking of the endogenous peroxidase with 3% H2O2 for 15 min. To reduce nonspecific binding of the primary antibody, tissues were blocked with blocking solution (CAS Block, Invitrogen, San Diego, CA), followed by incubation with the primary antibody overnight at 4 °C: Rb polyclonal anti WWOX [70 (link)] (1:8,000); Rb anti CK14 (ab181595, 1:2000); Rb anti ER (1:350, Sc-543); Rb anti p53 (1:400, NCLP53CM5p). Sections were washed 3 times with TBST and incubated with secondary HRP anti-rabbit IgG antibody for 30 min. After additional washes using TBST, the reaction was then performed using a DAB peroxidase kit (Vector Laboratories, SK-4100, Mowry Ave Newark, United State), followed by hematoxylin stain for 45 s as a counterstain.

Bidany-Mizrahi T., Shweiki A., Maroun K., Abu-Tair L., Mali B, & Aqeilan R.I. (2024). Unveiling the relationship between WWOX and BRCA1 in mammary tumorigenicity and in DNA repair pathway selection. Cell Death Discovery, 10, 145.

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Immunohistochemical Analysis of Sox17 and Wnt7a

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Slides of paraffin-embedded uteri were deparaffinzed, rehydrated, and incubated in antigen retrieval buffer prior to overnight incubation with 1ug/ml primary antibody Sox17 goat (R&D Systems; AF1924), 5ug/ml Wnt7a goat (R&D Systems; AF3008), or normal goat IgG control antibody (R&D Systems; AB-108-C). Slides were subsequently incubated in ImmPRESS Anti-Goat Ig (Vector Laboratories; MP-7405), labeled using the Vector NovaRED Peroxidase Substrate Kit (Vector Laboratories; SK-4800) or DAB Peroxidase Kit (Vector Laboratories; SK-4100) (in the case of Wnt7a-labeled tissue), and counterstained with Hematoxylin QS (Vector Laboratories; H3404). Goat IgG control primary antibody resulted in no cell labeling (Fig. S1). Detailed protocol is available in the Supplemental Materials and Methods.

Guimarães-Young A., Neff T., Dupuy A.J, & Goodheart M.J. (2016). Conditional deletion of Sox17 reveals complex effects on uterine adenogenesis and function. Developmental biology, 414(2), 219-227.

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