Ovarian tumor tissues collected before and after chemotherapy were stained for the expression of RAD6 and SOX2 proteins by immunohistochemistry. Antigen retrieval was carried out by heating the tissue specimens in 10mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity was inhibited by incubating the sections in 3% hydrogen peroxidase for 10 min. Tissue sections were incubated with specific primary antibodies, followed by a specific biotinylated secondary antibody, and then conjugated HRP streptavidin and DAB (3,3′-Diaminobenzidine) chromogen (Vector Laboratories, Burlingame, CA) and tissues were counterstained with hematoxylin (Vector Laboratories). Stained sections were analyzed by Zeiss Axioscope 2 microscope and images captured by AxioCam camera. DAB intensity was analyzed by Fiji Image J software. This process was carried out without any knowledge of the identity of each tissue sample to prevent bias in scoring the samples.
3 3 diaminobenzidine dab chromogen
Manufactured by Vector Laboratories
Sourced in Canada
3,3′-diaminobenzidine (DAB) chromogen is a widely used reagent in immunohistochemistry and in situ hybridization techniques. It serves as a chromogenic substrate, producing a brown insoluble precipitate upon enzymatic reaction.
Automatically generated - may contain errors
Lab products found in correlation
Hematoxylin, by Vector Laboratories (3 mentions)
Streptavidin hrp, by Vector Laboratories (2 mentions)
Axioscope 2 microscope, by Zeiss (2 mentions)
Axiocam camera, by Zeiss (2 mentions)
Vectastain universal abc kit, by Vector Laboratories (1 mentions)
Hematoxylin, by Thermo Fisher Scientific (1 mentions)
Deadend colorimetric apoptosis detection system, by Promega (1 mentions)
Envision system hrp, by Agilent Technologies (1 mentions)
Ki 67 antibody, by Agilent Technologies (1 mentions)
Permount, by Thermo Fisher Scientific (1 mentions)
Trypsin edta solution, by Thermo Fisher Scientific (1 mentions)
Streptavidin horseradish peroxidase, by Vector Laboratories (1 mentions)
Polyclonal rabbit anti human vwf antibody, by Agilent Technologies (1 mentions)
Vectamount, by Vector Laboratories (1 mentions)
Ab186834, by Abcam (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Fast red, by Merck Group (1 mentions)
Tcmk 1 cells, by Thermo Fisher Scientific (1 mentions)
Sa 5100, by Vector Laboratories (1 mentions)
Ba 1000, by Vector Laboratories (1 mentions)
9 protocols using 3 3 diaminobenzidine dab chromogen
1
Immunohistochemical Analysis of RAD6 and SOX2 in Ovarian Tumors
2
Immunohistochemical Analysis of RAD6 and SOX2 in Ovarian Tumors
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Ovarian tumor tissues collected before and after chemotherapy were stained for the expression of RAD6 and SOX2 proteins by immunohistochemistry. Antigen retrieval was carried out by heating the tissue specimens in 10mM sodium citrate buffer, pH 6.0. Endogenous peroxidase activity was inhibited by incubating the sections in 3% hydrogen peroxidase for 10 min. Tissue sections were incubated with specific primary antibodies, followed by a specific biotinylated secondary antibody, and then conjugated HRP streptavidin and DAB (3,3′-Diaminobenzidine) chromogen (Vector Laboratories, Burlingame, CA) and tissues were counterstained with hematoxylin (Vector Laboratories). Stained sections were analyzed by Zeiss Axioscope 2 microscope and images captured by AxioCam camera. DAB intensity was analyzed by Fiji Image J software. This process was carried out without any knowledge of the identity of each tissue sample to prevent bias in scoring the samples.
3
Immunohistochemistry of Lung Disease Tissue
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The lung disease spectrum tissue microarray slide LC1201(Biomax, Cheltenham, UK) was used in this project. IHC was performed using the VECTASTAIN® Universal ABC Kit (Vector Laboratories Inc., Upper Heyford, UK). The TMA slide was dewaxed and rehydrated and treated with 30% hydrogen peroxide (H2O2) followed by microwaving for 20 min. After cooling, the slide was blocked with horse serum (1–2 drops) in 5 mL of 1× OptiMax Wash Buffer (BioGenex, Fremont, CA, USA) for 2 h. After washing, the slide was incubated with primary antibody (10 μg/mL) overnight at 4 °C. Following incubation with the corresponding secondary antibody, VECTASTAIN® Universal ABC complex (Vector Laboratories Inc.) was added, then 3,3′-Diaminobenzidine (DAB) chromogen (Vector Laboratories Inc.) and the slide incubated in the dark. The target protein was indicated by brown colouration.
4
Immunohistochemistry of hCD45 in Mouse
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Deparaffinized and hydrated 10 μm ME sections were heated in citrate solution (pH 6.0) for antigen retrieval. After quenching endogenous peroxidase, sections were incubated with anti-hCD45 antibody (DAKO) for 24 h. Immunoreaction was detected by horseradish peroxidase-labeled ImmPRESS anti-mouse/rabbit secondary antibody (Vector Laboratories) developed with 3,3′-diaminobenzidine (DAB) chromogen (Vector Laboratories) and counterstained with hematoxylin (Vector Laboratories). Immunostained slides were imaged with an FSX100 microscope and exposure-matched pictures from humanized vs. NSG and BALB/c control mice compared.
5
Immunohistochemical Analysis of Tissue Sections
6
Immunohistochemical Analysis of HB-EGF Expression
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Deparaffinized and hydrated sections of control rat MEs, and Poly(I:C)-injected MEs harvested 2 days later, were heated in citrate solution (pH 6.0) for antigen retrieval. After quenching endogenous peroxidases, sections were incubated with anti-HB-EGF antibody (rabbit polyclonal, 1:200, Bioss) that recognizes both forms of the factor for 24 hours. Immunoreaction was detected by ImmPRESS mouse/anti-rabbit secondary antibody (Vector Laboratories) developed with 3, 3’-diaminobenzidine (DAB) chromogen (Vector Laboratories). Slides were counterstained only with hematoxylin.
7
Quantifying Angiogenesis in Tissue Scaffolds
8
Oxidative Stress in Kidney Epithelial Cells
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TCMK-1 cells (American Type Culture Collection, Manassas, USA), mouse kidney epithelial cell line, were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F-12 medium (Gibco, Carlsbad, USA) with 10% (v/v) fetal calf serum (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin G, and 100 mg/ml streptomycin (Sigma, Dorset, UK) at 37°C in a 5% CO2 humidified atmosphere.
TCMK-1 cells were cultured in serum-free DMEM/F-12 medium (Gibco, Carlsbad, USA) and stimulated with 200 μM H2O2 for 24 h. The cells were then fixed and subjected to ISEL and developed with 3,3′-diaminobenzidine (DAB) chromogen (Vector). Antiproperdin antibody (ab186834, Abcam) diluted 1:100 was applied followed by biotinylated goat–antirabbit IgG (1:300, BA-1000, Vector) and alkaline phosphatase streptavidin (1:200, SA-5100, Vector). Binding was detected by Fast Red (Sigma) and hematoxylin counterstaining. For the negative control, the primary antibody was substituted with normal rabbit IgG of same species (Merck Millipore) at same protein concentration.
TCMK-1 cells were cultured in serum-free DMEM/F-12 medium (Gibco, Carlsbad, USA) and stimulated with 200 μM H2O2 for 24 h. The cells were then fixed and subjected to ISEL and developed with 3,3′-diaminobenzidine (DAB) chromogen (Vector). Antiproperdin antibody (ab186834, Abcam) diluted 1:100 was applied followed by biotinylated goat–antirabbit IgG (1:300, BA-1000, Vector) and alkaline phosphatase streptavidin (1:200, SA-5100, Vector). Binding was detected by Fast Red (Sigma) and hematoxylin counterstaining. For the negative control, the primary antibody was substituted with normal rabbit IgG of same species (Merck Millipore) at same protein concentration.
9
Immunohistochemical Profiling of Trk Receptors
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Immunohistochemical staining was performed by a horseradish-peroxidase (HRP) technique using either a DAKO Autostainer (DAKO, Hamburg, Germany) or Ventana Discovery XT (Ventana Inc., Tucson, AZ) according to the manufacturer's recommendation. Paraffin-embedded tissue sections cut from the TMA were mounted on slides and treated as follows: (1) TrkA (Millipore, Billerica, MA) (clone MABN681)-1:200 dilution for 30 min, HRP flex secondary, and no antigen retrieval; (2) TrkB Millipore (clone AB9872)-1:600 dilution for 120 min, HRP flex secondary, and no antigen retrieval; (3) TrkC (Abcam, Cambridge, MA) (clone ab33656)-1:200 dilution for 120 min and anti-goat secondary with no antigen retrieval; (4) Pan-Trk (Abcam) (clone ab79770)-1:50 dilution for 15 min, Flex HRP secondary, and no antigen retrieval; and (5) NEUROD1 (Abcam) (clone ab60704)-monoclonal mouse, 1:800 dilution for 60 min, HRP flex secondary, and high pH (7) antigen retrieval. TrkA, B, and C and Pan-Trk antibodies were also double blocked with Avidin-Biotin and Background Sniper (Biocare Medical, Concord, CA) to reduce background. All antibodies were then visualized with 3,3′-diaminobenzidine (DAB) chromogen (Vector Laboratories, Burlingame, CA), and hematoxylin was used for counterstaining.
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