Guazatine, N,N′-dimethylthiourea (DMTU), nitroblue tetra zolium (NBT), 3, 3-diaminobenzidine hydrochloride (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), Spm, Spd, Put, 4-amino antipyrine, N,N′-dimethylaniline, and horseradish peroxidase were purchased from Sigma-Aldrich. Water used was always doubly distilled.
3 3 diaminobenzidine hydrochloride
Manufactured by Merck Group
Sourced in United States
3,3′-diaminobenzidine hydrochloride is a chemical compound used as a laboratory reagent. It is a chromogenic substrate that can be used in various immunohistochemical and enzymatic applications for the detection and visualization of target molecules in biological samples.
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Lab products found in correlation
Nitroblue tetrazolium, by Merck Group (2 mentions)
3 3 5 5 tetramethylbenzidine (tmb), by Merck Group (2 mentions)
N n dimethylthiourea, by Merck Group (1 mentions)
4 aminoantipyrine, by Merck Group (1 mentions)
N n dimethylaniline, by Merck Group (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Hematoxylin, by Beyotime (1 mentions)
Paraformaldehyde (pfa), by Merck Group (1 mentions)
Diniconazole, by Merck Group (1 mentions)
Fluridone, by Merck Group (1 mentions)
2 3 5 triphenyltetrazolium chloride, by Merck Group (1 mentions)
Coumarin, by Merck Group (1 mentions)
Streptavidin biotin peroxidase complex method, by Vector Laboratories (1 mentions)
Vectastain elite abc kit, by Vector Laboratories (1 mentions)
Cryostat, by Leica camera (1 mentions)
Phosphate buffered saline (pbs), by Merck Group (1 mentions)
Cytoseal 60, by Thermo Fisher Scientific (1 mentions)
Rabbit anti rat, by Vector Laboratories (1 mentions)
Sm2000r, by Leica Microsystems (1 mentions)
Mouse anti gfap, by Thermo Fisher Scientific (1 mentions)
9 protocols using 3 3 diaminobenzidine hydrochloride
1
Enzymatic Assays for Polyamines
2
Immunohistochemical Analysis of Signaling Proteins
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Tissues were fixed with parafor-maldehyde (4%; Sigma-Aldrich), then embedded in paraffin and sectioned. Sections were deparaffinized in xylol (Beyotime Institute of Biotechnology) and rehydrated in a graded ethanol series (China National Medicines, Shanghai, China). Following antigen retrieval by microwave heating (600 W; 15 min; P70F23P-G5; Galanz, Guangdong, China), the sections were incubated in non-immune serum (Santa Cruz Biotechnology, Inc.) for 30–60 min at room temperature and then blotted with the indicated primary antibody against p-Akt, p-HDM2 (Ser166), p53 or PTEN, overnight at 4°C. The next day, the sections were incubated in rabbit HRP-conjugated secondary antibody for 1 h at room temperature.
Finally, the sections were visualized with 3,3′-diamino-benzidine hydrochloride (Sigma-Aldrich) and counterstained with hematoxylin (Beyotime Institute of Biotechnology).
Finally, the sections were visualized with 3,3′-diamino-benzidine hydrochloride (Sigma-Aldrich) and counterstained with hematoxylin (Beyotime Institute of Biotechnology).
3
Phytochemical Screening and Analysis
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Coumarin, fluridone, diniconazole, ABA, nitroblue tetrazolium (NBT), 3,3′-diaminobenzidine hydrochloride (DAB), 3,3′,5,5′-tetramethylbenzidine (TMB), and 2,3,5-triphenyl tetrazolium chloride (TTC) were purchased from Sigma-Aldrich (USA). Water was doubly distilled before use.
4
Endometrial Immune Profiling in Recurrent Pregnancy Loss
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Endometrial immune-phenotype characterization in the women with RPL was obtained with endometrial parallel sections (3 μm) cut, deparaffined, and incubated overnight at 4 °C with primary antibodies against CD138 (plasma cell marker), CD20 (B lymphocyte marker), CD3, and TIA1 (T-cell markers), and progesterone and estrogen receptors. The peroxidase ABC method (Vector Laboratories, Burlingame, CA, USA) was performed for 1 h at room temperature, and 3′,3′ diaminobenzidine hydrochloride (Sigma, St Louis, MO, USA) was used as the chromogen.
5
Immunohistochemical Staining for Trop-2 Protein
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Immunohistochemical staining was performed in 4-μm-thick paraffin-embedded tissue sections. Sections were deparaffinized and rehydrated with xylene and a graded series of ethyl alcohols (from 100 % to 50 %) before incubation with 3 % hydrogen peroxide for 30 min to block endogenous peroxidase activity. Non-specific antibody binding was blocked with normal goat serum diluted 1:75 (30 min at room temperature). Afterwards, sections were incubated overnight at 4 °C with anti-Trop-2 mouse monoclonal antibody (no. SC-376181, Santa Cruz Biotechnology, Dallas, TX, USA) diluted 1:200 in phosphate buffered saline (PBS). After washing in PBS, the antigen was visualized by the streptavidin-biotin-peroxidase complex method (Vector Laboratories, Burlingame, CA, USA) using a biotinylated goat anti-mouse secondary antibody diluted 1:200. 3’, 3’- diaminobenzidine hydrochloride (Sigma-Aldrich, St Louis, MO, USA) was used as the chromogen. Sections were counterstained with Mayer's haematoxylin, dehydrated, and mounted in Eukitt solution (Kindler GmbH and Co., Freiburg, Germany). Negative controls were obtained by replacing the primary or secondary antibody with PBS. Additional negative controls included isotype-matched immunoglobulins (no. SC-2025, Santa Cruz Biotechnology). Normal human skin was used as a positive control for Trop-2 staining [8 (link)].
6
Liver Cell Proliferation in Rats
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Livers from all rats were examined by BrdU immunohistochemistry. Serial sections (3 μm) were cut from paraffin-embedded liver tissues and mounted on poly-l-lysine-coated slides. Established procedures for immunohistochemical staining with the avidin-biotin-peroxidase complex (ABC) method were used. Paraffin sections were deparaffinized and rehydrated through graded alcohols. Endogenous peroxidase activity was blocked with 0.3% H2O2 in distilled water for 5 min, and then antigen retrieval was performed by microwaving at 98°C for 20 min in 0.01 M citrate buffer (pH 6.0). After blocking nonspecific binding with normal horse serum at 37°C for 30 min, sections were incubated with mouse monoclonal anti-BrdU antibody (Dako Japan, Kyoto, Japan) at 1:500 dilution overnight at 4°C. Immunoreactivity was detected using a Vectastain Elite ABC Kit (PK-6102; Vector Laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine hydrochloride (Sigma Chemical Co., St Louis, MO, USA) followed by counterstaining with Mayer’s hematoxylin. For BrdU immunohistochemistry, small intestine formalin-fixed paraffin-embedded sections were used as positive controls. A negative control was also included with each staining procedure by omitting the primary antibody.
At least 3,000 hepatocyte nuclei were counted in each liver; labeling indices were calculated as the percentage of cells positive for BrdU.
At least 3,000 hepatocyte nuclei were counted in each liver; labeling indices were calculated as the percentage of cells positive for BrdU.
7
Tyrosine Hydroxylase Immunohistochemistry in Rat OB
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Rats were killed with an overdose of ketamine/xylazine, and then the body circuit was perfused transcardially with 50 mL of 4 °C cold 0.9% saline, followed by 400 mL of 4% paraformaldehyde (in phosphate-buffered saline (PBS), 0.1 M, pH 7.4). The brains were immediately removed from the skull and postfixed overnight at 4 °C in 4% paraformaldehyde solution. Subsequently, brains were cryoprotected at 4 °C for 48 h in 20% sucrose solution and snap frozen in −50 °C cold isopentane. The brains were stored at −80 °C. Frontal 30 μm-thick brain slices were serially cut with a cryostat (Leica, Germany). Histological sections of the OB were immunohistochemically reacted with an antibody against tyrosine hydroxylase (TH). For this purpose, the sections were washed in PBS, endogenous peroxidases were blocked using 3% hydrogen peroxide solution, and non-specific binding sites were blocked using horse serum. Primary antibody incubation (monoclonal, anti-TH, mouse, 1:1000, Sigma-Aldrich, St. Louis, MO, USA) was performed overnight at 4 °C. Secondary antibody incubation (polyclonal, anti-mouse, horse, 1:200, Vector Laboratories, Burlingame, CA, USA) was also performed at 4 °C overnight. Immunohistochemical labeling was visualized using a standardized 3,3′diaminobenzidine hydrochloride (10 mg/100 mL phosphate-buffered saline, Sigma-Aldrich, St. Louis, MO, USA) procedure.
8
Immunohistochemistry Protocol for GFAP, CD68, and c-Fos
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Fixed hemibrains were sectioned sagittally in 35 μm sections on a freezing microtome (SM2000R, Leica Microsystems). Free-floating sections were blocked in goat serum and incubated in primary antibody overnight at 4°C, followed by incubation in secondary antibody for 1 h. For GFAP staining, 1:500 mouse anti-GFAP (Thermo Scientific) was the primary antibody, and 1:500 goat anti-mouse (Vector Laboratories) the secondary antibody. For CD68 staining, 1:800 rat anti-CD68 (AbD Serotec) was the primary antibody, and 1:500 rabbit anti-rat (Vector Laboratories) was the secondary antibody. For c-Fos staining, 1:10,000 rabbit anti-c-Fos (Ab-5, Calbiochem) was the primary antibody, and 1:200 goat anti-rabbit (Vector Laboratories) was the biotinylated secondary antibody. After exposure to the secondary antibody, sections were incubated in avidin–biotin complex (ABC Reagent, Vector Laboratories) reagent and developed in 3,3' diaminobenzidine hydrochloride (Sigma-Aldrich) solution. Stained sections were mounted onto slides with 1% gelatin (Bio-Rad Laboratories), dried overnight, and then dipped in xylene (EMD Chemicals) for 5 min before coverslipping with Cytoseal 60 (Thermo Scientific).
9
Immunohistochemical Analysis of Aβ and Iba1
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Serial sections (4-µm thickness) cut from paraffin-embedded brain specimens were examined for expression of Aβ peptides and ionised calcium binding adaptor molecule 1 (Iba1) by immunohistochemical staining using an avidin-biotin-peroxidase complex (ABC) method. Antigen retrieval was performed for Aβ staining by microwaving sections at 98°C for 20 min in 0.01 M citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked with 3% H 2 O 2 in distilled water for 5 min. After blocking non-specific binding with goat serum at 37°C for 30 min, sections were incubated with rabbit polyclonal anti-β amyloid antibody (ab2539, Abcam, Cambridge, UK) at a dilution of 1:20, or rabbit polyclonal anti-Iba1 antibody (090-19741, Wako, Tokyo, Japan) at a dilution of 1:500 overnight at 4°C. Immunoreactivity was detected using a VECSTAIN Elite ABC Kit (PK-6101, Vector Laboratories, Burlingame, CA, USA) and 3,3′diaminobenzidine hydrochloride (Sigma Chemical Co., St Louis, MO, USA). Omission of the primary antibody served as the negative control, which was included with each staining procedure.
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