Bladder tissue was analyzed for signs of inflammation with standard HE staining. Free floating immunohistochemistry was performed for spinal cord c-fos experiments. Tissue was rinsed with 0.1 M PBS solution containing 0.1 % (v/v) bovine serum albumin and 0.3 % (v/v) Triton-X-100 (PBS-BT). Tissue was incubated overnight with a c-fos (4) antibody (c-fos (4) sc-52 rabbit-polyclonal Lot # L 1809, Santa Cruz®, Santa Cruz, USA) dissolved in PBS-BT at room temperature. We used 0.1 M PBS pH 7.3 for further rinsing steps. Tissue was incubated with a donkey-a-rabbit IgG biotin conjugated secondary antibody (Jackson Imm Research®, West Grove, USA) in PBS-BT for 90 min, following incubation with Vector ABC elite® (Vector, Burlingame, USA) for 90 min. Tissue was incubated using a DAB solution containing 10 μl DAB (Sigma-Aldrich, St Louis, USA) and 150 mg ammonium-nickel-sulfate dissolved in 50 ml 0.05 M Tris buffered saline for 10 min, followed by a 10 min incubation with the same DAB-nickel solution with the addition of perhydrol (5 μl in 25 ml DAB-nickel solution). Tissue was mounted on objects glasses coated with gelatin and dried overnight at 37 °C, then dehydrated with ethanol and xylene and mounted with Permount® (Thermo Fisher Scientific, Waltham, USA). Sections were analyzed by using binocular (Leica DMR®) microscope and processed with image J 1.41o® software.
3 3 diaminobenzidine (dab)
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom, Italy, China, Japan, Canada, Sao Tome and Principe, Denmark, France, Macao, Australia, Spain, Switzerland
3,3'-diaminobenzidine is a chemical compound commonly used as a chromogenic substrate in various laboratory techniques, such as immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It is a sensitive and specific reagent that can be used to detect the presence of target proteins or enzymes in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (38 mentions)
Vectastain abc kit, by Vector Laboratories (36 mentions)
Vectastain elite abc kit, by Vector Laboratories (34 mentions)
Abc kit, by Vector Laboratories (28 mentions)
Avidin biotin peroxidase complex, by Vector Laboratories (22 mentions)
Biotinylated secondary antibody, by Vector Laboratories (22 mentions)
Hematoxylin, by Merck Group (21 mentions)
Triton x 100, by Merck Group (17 mentions)
Hydrogen peroxide, by Merck Group (17 mentions)
Permount, by Thermo Fisher Scientific (15 mentions)
Biotinylated goat anti rabbit igg, by Vector Laboratories (14 mentions)
Entellan, by Merck Group (14 mentions)
Avidin biotin complex, by Vector Laboratories (13 mentions)
Abc reagent, by Vector Laboratories (13 mentions)
Vectastain abc reagent, by Vector Laboratories (13 mentions)
Cytochrome c, by Merck Group (12 mentions)
Bx51 microscope, by Olympus (10 mentions)
Cellsens software, by Olympus (9 mentions)
Nitrocellulose membrane, by Merck Group (9 mentions)
Biotinylated anti rabbit secondary antibody, by Vector Laboratories (9 mentions)
1 474 protocols using 3 3 diaminobenzidine (dab)
1
Immunohistochemical Analysis of c-Fos Expression
2
GUS and DAB Staining of Plant Tissues
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GUS staining was conducted using β-Galactosidase Reporter Gene Staining Kit (Beijing Leagene Biotech. Co., Ltd., Cat No./ID: DP0013, Beijing, China). Samples were placed into GUS staining solution and incubated at 37°C overnight. The tissues then were placed in 95% ethanol until chlorophyll was washed out, and samples were photographed. The tissues were stored in Formaldehyde-Acetic Acid-Ethanol (FAA) Fix Solution (Wuhan Servicebio Technology Co., Ltd., Cat No./ID: G1103-500 ML, Wuhan, China). For H2O2 visualization with DAB staining, leaves were put in 1mg/ml DAB (3, 3'-Diaminobenzidine tetrahydrochloride hydrate; Sigma-Aldrich, Cat No./ID: D5637-1G, Darmstadt, Germany) and vacuum infiltrated until DAB solution was taken up, after which the leaves were incubated in DAB staining solution for 16h in the dark. The leaves were subsequently transferred to 95% ethanol for 24h to remove chlorophyll. Afterward, leaves were photographed (Kissoudis, 2016 ).
3
Immunohistochemical Analysis of Ki67 and PRKCA
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Excised tumors were fixed with 4% paraformaldehyde, and embedded in paraffin and sectioned at 4 μm thickness. Hydrogen peroxide at 3% was used to block the endogenous peroxidase activity for 20 min. Antigen was exposed in 10 mM citrate buffer (pH 6.0) by heat repair at 98°C for 2 min. Sections were incubated with primary anti-ki67 (ab92742, Abcam) overnight at 4°C and secondary antibody (ab6721, Abcam) for 20 min at room temperature. Immunoreactivity was visualized using 3,3′-diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA) and the sections were counterstained with hematoxylin.
Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 20 min. Antigen was exposed by heat repair in 10 mM citrate buffer (pH 6.0) at 98°C for 2 min. The sections were treated and incubated with primary anti-PRKCA (1:100 dilution; Abcam, Cambridge, UK) for 16 h at 4°C and horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 20 min at room temperature. Immunoreactivity was visualized using 3,3ʹ-diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA) and sections were counterstained with hematoxylin and observed under a microscope.
Endogenous peroxidase activity was blocked by incubation in 3% hydrogen peroxide for 20 min. Antigen was exposed by heat repair in 10 mM citrate buffer (pH 6.0) at 98°C for 2 min. The sections were treated and incubated with primary anti-PRKCA (1:100 dilution; Abcam, Cambridge, UK) for 16 h at 4°C and horseradish peroxidase (HRP)-conjugated secondary antibody (Santa Cruz Biotechnology, CA, USA) for 20 min at room temperature. Immunoreactivity was visualized using 3,3ʹ-diaminobenzidine (Sigma Chemical Co., St. Louis, MO, USA) and sections were counterstained with hematoxylin and observed under a microscope.
4
PHA-L Immunolabeling Protocol
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For PHA-L immunolabeling, sections were preincubated for 1 h in PBS containing 0.4% Triton X-100 and 1% normal goat serum (NGS), then incubated overnight with the primary antibody (rabbit anti-PHA-L, 1/2,000, AB_2313686, Vector Laboratories, Burlingame, CA, USA). After rinsing for 30 min with PBS, sections were incubated for 1 h with the secondary antibody (goat biotinylated anti-rabbit, 1/200, AB_2313606, Vector) in PBS containing 0.4% Triton X-100 and 1% NGS, then rinsed with PBS and incubated for 1 h in the avidin-biotin-horseradish-peroxidase solution (ABC Vectastain Elite Kit, Vector Laboratories). Sections were then processed in two series. The first series was stained (brown/orange) with 0.4% 3,3’-diaminobenzidine (DAB, Sigma-Aldrich, St Louis, MO, USA) in PBS supplemented with H2O2 at progressively increasing concentrations (0.00025%, 0.0005%, 0.001%, 0.002%, 0.004%). The second series was first rinsed with 0.1 M Tris-HCl (pH 7.4, 15 min) and then stained (black/gray) with 0.4% DAB (Sigma-Aldrich) + 2% ammonium nickel sulfate in Tris buffer supplemented with increasing concentrations of H2O2 as described above. After rinsing in respective buffer, sections on glass slides were cover-slipped with Eukitt (Sigma-Aldrich).
5
Visualizing Reactive Oxygen Species in Plants
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To visualize H2O2 localization, leaves from all the treatments were immersed in a 1% solution of 3,3'-diaminobenzidine (DAB) (Sigma-Aldrich, St. Louis, MO, USA) in Tris-HCl buffer (pH 6.5), vacuum-infiltrated for 5 min placed in closed vacuum jar attached with suction pump to apply and release vacuum. After vacuum infiltration leaves were incubated at room temperature (25°C) for 2–3 h in the absence of light. Leaves were illuminated until the appearance of brown spots characteristic of the reaction of DAB (Sigma-Aldrich, St. Louis, MO, USA) with H2O2 (hydrogen peroxide). Leaves were bleached by immersing in boiling ethanol to visualize the brown spots and were photographed with a digital camera (Nikon, Japan) at a default setting of 600 dpi.
For the visualization of O2−1, leaves were immersed in a 0.1% solution of nitro blue tetrazolium (NBT) (Sigma-Aldrich, St. Louis, MO, USA) in K-phosphate buffer (pH 6.4), containing 10 mM Na-azide (Sigma-Aldrich, St. Louis, MO, USA), and were vacuum-infiltrated for 5 min placed in closed vacuum jar attached with suction pump to apply and release vacuum. After vacuum infiltration leaves were illuminated until the appearance of dark blue spots (characteristic of blue formazan precipitate). After bleaching in boiling ethanol, the leaf samples were photographed as described above.
For the visualization of O2−1, leaves were immersed in a 0.1% solution of nitro blue tetrazolium (NBT) (Sigma-Aldrich, St. Louis, MO, USA) in K-phosphate buffer (pH 6.4), containing 10 mM Na-azide (Sigma-Aldrich, St. Louis, MO, USA), and were vacuum-infiltrated for 5 min placed in closed vacuum jar attached with suction pump to apply and release vacuum. After vacuum infiltration leaves were illuminated until the appearance of dark blue spots (characteristic of blue formazan precipitate). After bleaching in boiling ethanol, the leaf samples were photographed as described above.
6
Visualizing Plant Cell Death and Oxidative Stress
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For visualization of cell death, EE was gently removed and leaves were submerged in lactophenol trypan blue solution (5 mL of lactic acid, 10 mL of 50% [v/v] glycerol, 1 mg of trypan blue [Sigma], and 5 mL of phenol) at 28°C for 2-3 h. Hydrogen peroxide (H 2 O 2 ) accumulation was measured with 3,3-diaminobenzidine (DAB; Sigma). Leaves were submerged in a 1.0 mg mL -1 DAB solution and incubated in the dark at room temperature for 6-8 h. Superoxide radical (O 2 . -) was visualized with the sensitive dye nitroblue tetrazolium (NBT; Sigma). Leaves were submerged in a solution containing 0.02% NBT and 10 mM NaN 3 for 4 h at room temperature in the dark.
After each staining, leaves were destained in boiling 95% (v/v) ethanol. Microscope images were saved as TIFF files and processed for densitometric quantification with ImageJ version 1.64 (NIH).
After each staining, leaves were destained in boiling 95% (v/v) ethanol. Microscope images were saved as TIFF files and processed for densitometric quantification with ImageJ version 1.64 (NIH).
7
In Situ Visualization of Plant Oxidative Stress
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Treated cotyledons were detached and infiltrated under vacuum with diaminobenzidine tetrahydrochloride (DAB; Šašek et al., 2012b (link)) aqueous solution (10 mg·ml−1, Sigma–Aldrich), with DAB being solubilized in dimethylformamide. Cotyledons were kept in humid conditions in darkness at room temperature until reddish-brown staining appeared. Chlorophyll was removed using 96% EtOH, after which cotyledons were rehydrated and scanned.
8
Quantifying Cellular Oxidative Stress
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For the DCFH-DA staining assay, root tips from 7-day-old seedlings were incubated in darkness for 30 min in a solution containing phosphate-buffered saline (PBS; pH 6.0) and 50 μM DCFH-DA. They were then washed three times with PBS (pH 6.0) to remove the excess DCFH-DA. Fluorescence was detected with a Leica SP8 confocal microscope.
An Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) was used to measure H2O2 production as described by Shin et al.56 (link).
For DAB staining to detect H2O2, seven-day-old seedlings were incubated in 0.1 mg/mL DAB (Sigma-Aldrich Corp., St. Louis, MO) dissolved in ddH2O (pH 5.0) for 8 h, and then in 75% ethanol at 65 °C for 15 min; after three washes, the roots were photographed.
An Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Invitrogen) was used to measure H2O2 production as described by Shin et al.56 (link).
For DAB staining to detect H2O2, seven-day-old seedlings were incubated in 0.1 mg/mL DAB (Sigma-Aldrich Corp., St. Louis, MO) dissolved in ddH2O (pH 5.0) for 8 h, and then in 75% ethanol at 65 °C for 15 min; after three washes, the roots were photographed.
9
Immunohistochemical Analysis of Liver Markers
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Immunohistochemical studies were carried out for detection of caspase-3 and α-SMA expression on paraffin sections of liver of control and all treated groups using avidin-biotin peroxidase (DAB, Sigma Chemical Co.) according to method described by Hsu et al. (1981) (link). Tissue sections were incubated with a monoclonal antibody for caspase-3 and α-SMA (Dako Corp, Carpenteria, CA, USA) and reagents required for the avidin-biotin peroxidase (Vactastain ABC peroxidase kit, Vector Laboratories) method for the detection of the antigen–antibody complex. Each marker expression was visualized by the chromagen 3,3 -diaminobenzidine tetra hydrochloride (DAB, Sigma Chemical Co.).
10
Histochemical GUS Staining of Arabidopsis Transgenics
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The histochemical GUS staining of the homozygous T3 transgenic lines harboring proAtSIBP1::GUS was done as described [35 (link)]. For DAB (3, 3’-Diaminobenzidine tetrahydrochloride) staining, five Arabidopsis rosette leaves treated with NaCl were placed in 5 mL tubes supplemented with a 3 mL DAB staining solution (1 mg/mL DAB, Sigma-Aldrich, adjusted to pH 3.8 with HCl), then vacuumed for 5 min. After 6 h of incubation in the dark with gentle shaking, the leaves were immersed in a bleaching solution (ethanol, acetic acid, and glycerol = 3:1:1) and boiled for 20 min to decolorize the leaves (except for the deep brown polymerization product that was produced by the reaction of DAB with H2O2), and then the images were captured.
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