3 3 diaminobenzidine (dab)

Manufactured by Wuhan Boster Biological Technology

Sourced in China

3,3'-diaminobenzidine is a commonly used chromogenic substrate in immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It is a peroxidase substrate that produces a brown-colored precipitate upon oxidation, allowing for the visualization of target proteins or antigens in biological samples.

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Lab products found in correlation

Bovine serum albumin (bsa), by Merck Group (2 mentions) Hematoxylin, by Wuhan Boster Biological Technology (2 mentions) Caspase 3, by Merck Group (1 mentions) Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions) Ab8366, by Abcam (1 mentions) Ab24590, by Abcam (1 mentions) Ba1001, by Wuhan Boster Biological Technology (1 mentions) Image pro plus 6, by Media Cybernetics (1 mentions) Dimethylbenzene, by Sinopharm (1 mentions) Axiovision 4, by Zeiss (1 mentions) Monoclonal mouse anti α smooth muscle actin antibody, by Thermo Fisher Scientific (1 mentions) Horseradish peroxidase hrp conjugated rabbit anti mouse igg, by Cell Signaling Technology (1 mentions) Neutral balsam, by OriGene (1 mentions) Biotinylated goat anti rabbit igg, by Wuhan Boster Biological Technology (1 mentions) Rabbit anti factor 8, by Bioss Antibodies (1 mentions) Inverted microscope, by Olympus (1 mentions) Goat serum, by Beyotime (1 mentions) Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions) Image plus pro5, by Media Cybernetics (1 mentions) Ts100, by Nikon (1 mentions)

8 protocols using 3 3 diaminobenzidine (dab)

Apoptotic Protein Expression in Broiler Thymuses

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At 14, 28 and 42 days of age, five broilers in each group were humanely euthanized. Thymuses were taken for the detection of Bax, Bcl-2 and caspase-3 protein expression by the immunohistochemical methods (SABC) and stained with DAB as described by Wang et al [91 (link)]. Anti-Bax (BA0315), anti-Bcl-2 (BA0412) and anti-caspase-3 (BA0588), and DAB were purchased from Wuhan Boster Biological Technology Co., Ltd., China.
Images from five slices per thymus were taken 200 μm apart. Five visions per slice were randomly chosen for assessment of positive cells using image analysis software (JID801D). The average grayscale of the positive cells was automatically calculated. Immunoreactive intensity was expressed by average grayscale. Values < 160 was considered high, 160-170 medium and 170-180 low.

Cui W., Guo H, & Cui H. (2015). Vanadium toxicity in the thymic development. Oncotarget, 6(30), 28661-28677.

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Western Blot Analysis of Apoptosis Markers

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Cells were harvested and total protein extracts were prepared using the Total Protein Extraction kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China) according to manufacturer's protocol. Total proteins were separated using 12% SDS-PAGE under 80–90 V and 250 mA for 30 min, and then transferred to polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc.). Then, the membranes were washed with PBS-Tween 20 twice (5 min/wash), and blocked with 1% bovine serum albumin at room temperature (Sigma-Aldrich, Merck KGaA) for 1 h. Subsequently, the membranes were incubated with caspase-3 (cat. no. BA2142), Fas (cat. no. BA0048), FasL (cat. no. BA0049) and GAPDH (cat. no. BA2913) primary antibodies (1:500/600; Wuhan Boster Biological Technology, Ltd.) at 4°C overnight. After washing with PBS-Tween 20 twice (5 min/wash), the membranes were incubated with biotinylated goat anti-rabbit IgG secondary antibody (cat. no. BA1003; 1:40/50; Wuhan Boster Biological Technology, Ltd.) at 37°C for 30 min. After washing with PBS-Tween 20 three times (5 min/wash), the membranes were incubated with streptavidin-labeled horseradish peroxidase (1:40/50; Wuhan Boster Biological Technology, Ltd.) for 30 min, followed by washing with PBS-Tween 20 twice (5 min/wash). Then, the membranes were stained with DAB (1:20; Wuhan Boster Biological Technology, Ltd.).

Wang A., Wang M., Pang Q., Jia L., Zhao J., Chen M, & Zhao Y. (2018). Lily extracts inhibit the proliferation of gastric carcinoma SGC-7901 cells by affecting cell cycle progression and apoptosis via the upregulation of caspase-3 and Fas proteins, and the downregulation of FasL protein. Oncology Letters, 16(2), 1397-1404.

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Histological Evaluation of New Bone Formation

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After micro-CT scanning, samples were dehydrated and made transparent using dimethylbenzene (Sinopharm Chemical Reagent Co., Ltd.). They were then embedded in wax and sectioned into 6 µm coronal planes. Sections were stained with hematoxylin and eosin (H&E) and observed under a light microscope (Leica Microsystems GmbH, Wetzlar, Germany). Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA) was used to evaluate new bone formation at ×100 magnification in six randomly selected fields per section. Bone density was defined as the ratio of new bone area to total area. The border of the new bone and osteoid tissue was difficult to define, so osteoid tissue was not included in new bone calculations.
Immunohistochemistry was performed using antibodies specific for CD31 (1:200; ab24590; Abcam) and HIF-1α (1:100; ab8366; Abcam). In brief, these sections were rehydrated and incubated with primary antibodies at 4°C overnight, then incubated with biotinylated secondary IgGs (1:500; BA1001; Wuhan Boster Biological Technology, Ltd., Wuhan, China). Sections were treated with ABC complex and developed with 3,3′-diaminobenzidine (both Wuhan Boster Biological Technology, Ltd.), then stained with hematoxylin. All sections were consistently maintained in liquid, and sections incubated without primary antibodies were used as a control.

Zhu Z.H., Song W.Q., Zhang C.Q, & Yin J.M. (2016). Dimethyloxaloylglycine increases bone repair capacity of adipose-derived stem cells in the treatment of osteonecrosis of the femoral head. Experimental and Therapeutic Medicine, 12(5), 2843-2850.

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Immunocytochemical Identification of Smooth Muscle Cells

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ASMCs were identified by α-smooth muscle actin immunocytochemical staining, as previously described (18 (link),19 (link)). Briefly, ASMCs were seeded onto sterile glass coverslips and grown to 70% confluence. Cells were fixed in 4% paraformaldehyde for 20 min, and the sections were reacted with 3% H₂O₂ (Guangzhou Whiga Technology Co., Ltd.) After a rinse in phosphate-buffered saline (PBS), the sections were blocked with 2% bovine serum albumin (BSA; Guangzhou Whiga Technology Co., Ltd.) and incubated with monoclonal mouse anti-α-smooth muscle actin antibody (1:300; Thermo Fisher Scientific, Inc.) at 4°C overnight for immunolabeling. Subsequently, the sections were incubated with a secondary horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG (1:300; Cell Signaling Technology, Inc., Danvers, MA, USA) for 30 min at 37°C and then washed three times with PBS. The peroxidase activity was visualized by a color reaction using 3,3′-diaminobenzidine (1 ml; Wuhan Boster Biological Technology, Ltd., Wuhan, China) as the substrate. The slides were then counterstained with hematoxylin (Wuhan Boster Biological Technology, Ltd.), mounted and examined under a microscope (Olympus Corp., Tokyo, Japan) using AxioVision 4.1 software (Carl Zeiss Microscopy, LLC, Thornwood, NY, USA).

LIN X., YANG C., HUANG L., CHEN M., SHI J., OUYANG L., TANG T., ZHANG W., LI Y., LIANG R, & JIANG S. (2016). Upregulation of TRPM7 augments cell proliferation and interleukin-8 release in airway smooth muscle cells of rats exposed to cigarette smoke. Molecular Medicine Reports, 13(6), 4995-5004.

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Immunohistochemical Validation of Factor VIII Expression in EC-like Cells

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To validate the EC-like cells, Factor VIII expression was assessed using an immunohistochemistry assay as previously described (31 (link)). Cells were fixed using 4% paraformaldehyde for 20 min at room temperature, treated with 0.3% H₂O₂ for 5 min at room temperature, permeabilized using 0.5% Triton X-100 and blocked using goat serum (Beyotime Institute of Biotechnology, Haimen, China) for 10 min at room temperature. The cells were incubated with rabbit anti-Factor VIII (1:200; BIOSS, Beijing, China; no. bs-0434R) for 4 h at 37°C. Following incubation with biotinylated goat anti rabbit IgG (1:200; Wuhan Boster Biological Technology, Ltd., Wuhan, China; BA1006) for 30 min at room temperature, the cells were treated with a streptavidin-biotin complex for 20 min at 37°C. The final immunoreactions were conducted using 3,3′-Diaminobenzidine for 5 min at room temperature according to the manufacturer's protocol (Wuhan Boster Biological Technology, Ltd.). Finally, the slides were mounted with neutral balsam (Origene Technologies, Inc., Beijing, China) and observed under an inverted microscope at ×200 magnification (Olympus Corp., Tokyo, Japan).

Li Z., Zhang S., Cao L., Li W., Ye Y.C., Shi Z.X., Wang Z.R., Sun L.X., Wang J.W., Jia L.T, & Wang W. (2017). Tanshinone IIA and Astragaloside IV promote the angiogenesis of mesenchymal stem cell-derived endothelial cell-like cells via upregulation of Cx37, Cx40 and Cx43. Experimental and Therapeutic Medicine, 15(2), 1847-1854.

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Immunohistochemical Analysis of CD68

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The paraffin-embedded sections were deparaffinized and rehydrated. They were then processed for antigen retrieval in a microwave oven. The sections were then rinsed in PBS containing 0.1% Tween-20 (PBS-T) and blocked in 3% peroxide-methanol at room temperature to inhibit endogenous peroxidase activity. The sections were blocked in 3% bovine serum albumin (Invitrogen; Thermo Fisher Scientific, Inc.), for 1 h at room temperature and then incubated with primary antibodies for CD68 (dilution, 1:200) overnight at 4°C. Following this, the sections were then incubated with HRP-conjugated anti-rabbit secondary antibody (dilution, 1:100) for 50 min at room temperature, color was developed using 3,3-diaminobenzidine (Wuhan Boster Biological Technology, Ltd.). After the sections were counterstained with hematoxylin (Wuhan Boster Biological Technology, Ltd.), the slides were sequentially washed through an alcohol dehydration series and mounted using neutral gum. The slides were examined using the inverted optical microscope (BX51; Olympus Corporation), The sizes of the stained areas were calculated using Image-Plus Pro5.0 (Media Cybernetics, Inc.).

Shi H., Liang M., Chen W., Sun X., Wang X., Li C., Yang Y., Yang Z, & Zeng W. (2017). Human induced pluripotent stem cell-derived mesenchymal stem cells alleviate atherosclerosis by modulating inflammatory responses. Molecular Medicine Reports, 17(1), 1461-1468.

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Immunohistochemical Analysis of SDF-1 and CXCR4

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NP tissues were fixed in 4% paraformaldehyde for 24 h, embedded in paraffin and cut serially at 5 μm for IHC staining. The IHC staining procedure was performed using a streptavidin-peroxidase immunohistochemical kit (Wuhan Boster Biological Technology, Ltd., Wuhan, China) according to the manufacturer's protocol. Briefly, the tissue sections were incubated with 0.125% trypsin for 30 min at 37°C for antigen retrieval and then incubated with primary rabbit monoclonal antibodies against SDF-1 (1:100; Abcam, Cambridge, UK) and CXCR4 (1:100; Abcam; cat. no. ab124824) overnight at 4°C to stain target protein expression, and immunoglobulin G (Wuhan Boster Biological Technology, Ltd) was used as a negative control. Finally, sections were incubated in 3–3′-diaminoben-zidine (Wuhan Boster Biological Technology, Ltd) to visualize immunoreactivity. Positively stained cells in three different areas were counted under a light microscope (Nikon TS100; Nikon Corporation, Tokyo, Japan).

LIU Z., MA C., SHEN J., WANG D., HAO J, & HU Z. (2016). SDF-1/CXCR4 axis induces apoptosis of human degenerative nucleus pulposus cells via the NF-κB pathway. Molecular Medicine Reports, 14(1), 783-789.

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Immunohistochemical Analysis of ZO-1 Expression in Rat Brain

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A total of 30 rats (10/group) were anesthetized with 10% chloral hydrate (400 mg/kg) and transcardially perfused with 0.9% saline followed by 4% paraformaldehyde (from the Pharmacy of Xiangya Hospital). Following sacrificed by decapitation, the brains were removed and stored in 4% paraformaldehyde until processing.
Frozen sections (30-µm) of brain tissue were brought to room temperature and incubated in 3% H₂O₂ for 15 min. After washing three times in phosphate-buffered saline for 5 min each, nonspecific binding was blocked with 5% bovine serum albumin (Sigma Aldrich) for 1 h at 37°C. Immunostaining was performed using rabbit anti-ZO-1 polyclonal antibody (1:400; cat. no. sc-5562; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight, followed by staining with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:200; cat. no. BA1054; Wuhan Boster Biological Technology, Ltd., Wuhan, China) for 120 min. Immunoreactivity was visualized using 3,3-diaminobenzidine (Wuhan Boster Biological Technology, Ltd.) under a light microscope (Zeiss Primo Star; Carl Zeiss AG, Oberkochen, Germany). For the image analysis, 10 microscopic fields were selected randomly from each group and the integrated optical densities (IODs) were measured using Image-Pro Plus 5.0 software (Media Cybernetics, Inc., Rockville, MD, USA).

WANG Y., PENG F., XIE G., CHEN Z.Q., LI H.G., TANG T, & LUO J.K. (2016). Rhubarb attenuates blood-brain barrier disruption via increased zonula occludens-1 expression in a rat model of intracerebral hemorrhage. Experimental and Therapeutic Medicine, 12(1), 250-256.

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