3 30 diaminobenzidine tetrahydrochloride

Manufactured by Merck Group

3,30-diaminobenzidine tetrahydrochloride is a chemical compound used in various laboratory applications. It serves as a chromogenic substrate for the detection and visualization of certain enzymes, particularly peroxidases, during immunohistochemistry and other analytical techniques. The compound undergoes an oxidation reaction in the presence of peroxidase, resulting in the formation of a brown, insoluble precipitate that can be observed under a microscope.

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Lab products found in correlation

Microscope slide scanner, by 3DHISTECH (2 mentions) Lsab system hrp kit, by Agilent Technologies (2 mentions) Avidin biotin complex system, by Vector Laboratories (2 mentions) Pk 6101, by Vector Laboratories (2 mentions) Abc elite kit, by Vector Laboratories (2 mentions) Anti cathepsin k, by Santa Cruz Biotechnology (1 mentions) Inverted microscope, by Nikon (1 mentions) Optiphot 2, by Nikon (1 mentions) Anti bsp, by Santa Cruz Biotechnology (1 mentions) Anti oc, by Santa Cruz Biotechnology (1 mentions) Anti nfatc1, by Santa Cruz Biotechnology (1 mentions) Biotin conjugated anti rabbit igg antibody, by Vector Laboratories (1 mentions) Avidin biotin peroxidase complex reagent, by Vector Laboratories (1 mentions) Streptavidin horseradish peroxidase, by BD (1 mentions)

9 protocols using 3 30 diaminobenzidine tetrahydrochloride

Immunohistochemical Detection of MIF

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Paraffin sections were deparaffinized in three solutions of xylene and rehydrated in a graded series of ethanol solutions. For antigen retrieval, slides were placed in 0.01 M citrate buffer at pH 6.0 and heated in a steamer for 30 min. Endogenous peroxidases were quenched by incubating the samples with 3% hydrogen peroxide for 20 min at room temperature. Sections were incubated overnight at 4°C using a 1:50 dilution of the primary antibody against MIF (Santa-Cruz Biotechnology Inc.). Sections were then incubated for 30 min with a biotinylated secondary antibody (LSAB system HRP kit; DakoCytomation, Glostrup, Denmark), rinsed in PBS, and incubated for 30 min with a streptavidin-peroxidase conjugate (LSAB; DakoCytomation). The reaction was developed for 5 min using 3, 30-diaminobenzidine tetrahydrochloride (Sigma-Aldrich; Merck KGaA). Slides were counterstained in hematoxylin, dehydrated, and covered with a cover slip. Negative and positive controls were simultaneously analyzed. Positive controls comprised mammary tissues. The slides were captured using a microscope slide scanner (3D-HISTECH Ltd., Budapest, Hungary).

Sohn H.M., Kim B., Park M., Ko Y.J., Moon Y.H., Sun J.M., Jeong B.C., Kim Y.W, & Lim W. (2019). Effect of CD133 overexpression on bone metastasis in prostate cancer cell line LNCaP. Oncology Letters, 18(2), 1189-1198.

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Immunohistochemical Analysis of Cathepsin K

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Paraffin sections were deparaffinized in three xylene washes and rehydrated in graded ethanol solutions. For antigen retrieval, the slides were placed in 0.01 M citrate buffer (pH 6.0) and heated in a steamer for 30 min. Endogenous peroxidases were quenched by incubating the samples with 3% hydrogen peroxide for 20 min at room temperature. The sections were incubated overnight at 4°C using 1:50 anti‐cathepsin K (Santa‑Cruz Biotechnology Inc.). Sections were then incubated for 30 min with biotinylated secondary antibody (LSAB system HRP kit; Dako Cytomation, Glostrup, Denmark), rinsed in PBS, and incubated for 30 min with a streptavidin‑peroxidase conjugate (LSAB; DakoCytomation). The reaction was developed for 5 min using 3,30‑diaminobenzidine tetrahydrochloride (Sigma‑Aldrich; Merck KGaA). The slides were counterstained in hematoxylin, dehydrated, and coverslipped. Negative and positive controls were simultaneously analyzed. The positive controls were mammary tissues. The slides were imaged using an inverted microscope (Nikon).

Ko Y.J., Sohn H.M., Jang Y., Park M., Kim B., Kim B., Park J., Hyun H., Jeong B., Hong C, & Lim W. (2021). A novel modified RANKL variant can prevent osteoporosis by acting as a vaccine and an inhibitor. Clinical and Translational Medicine, 11(3), e368.

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Visualizing Nuclear c-Fos Expression via DAB Staining

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We used DAB staining to better visualize nuclear cfos expression over a relatively large area. 30 minutes after i.p. dexmedetomidine (50, 100, 400 μg/kg), or saline (0.9%) administration, experimental mice were anesthetized, perfused transcardially, and free-floating sections processed for immunohistochemistry with rabbit antibody to Fos^{29 (link)} (1:20,000, Ab-5, Calbiochem) after blocking endogenous peroxidase with 0.3% H₂O₂ in PBS.The primary antiserum was localized using a variation of the avidin-biotin complex system (Vector Laboratories). In brief, sections were incubated for 120 minutes at 22–25 °C in a solution of biotinylated goat antibody to rabbit IgG (PK-6101, Vector Laboratories) and then placed in the mixed avidin-biotin horseradish peroxidase complex solution (ABC Elite Kit, Vector Laboratories) for 60 minutes. The peroxidase complex was visualized by a 4 to 5-minute exposure to chromogen solution (0.05% 3,30-diaminobenzidine tetrahydrochloride (wt/vol, Sigma-Aldrich), 0.4 mg/ml nickel ammonium sulfate to produce a blue-black product. The reaction was stopped by washing in distilled water and PBS. Sections were dehydrated and cover-slipped with quick mounting medium (Eukitt, Fluka Analytical).

Zhang Z., Ferretti V., Güntan İ., Moro A., Steinberg E.A., Ye Z., Zecharia A.Y., Yu X., Vyssotski A.L., Brickley S.G., Yustos R., Pillidge Z.E., Harding E.C., Wisden W, & Franks N.P. (2015). Neuronal ensembles sufficient for recovery sleep and the sedative actions of α2 adrenergic agonists. Nature neuroscience, 18(4), 553-561.

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Visualizing Nuclear c-Fos Expression via DAB Staining

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Quantifying GAL2 Receptor Changes in Rat Brain

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At 4, 6, 8, 10, 12, and 14 days after injection of siRNA GAL₂ receptor, two rats per day were perfused transcardially. The brains were then dissected and serial, coronal free-floating sections (30 μm thick) were collected. The control group received an injection of Accel siRNA Delivery Media (vehicle). The sections were incubated with a primary antibody rabbit anti-GAL₂ receptor (Alomone Lab, 1/250) overnight at 4ºC. An hour incubation with biotinylated, specific secondary antibodies (1:200; Vector Labs Inc) was performed at room temperature. The chromogen used was 0.03% 3-30-diaminobenzidine tetrahydrochloride (Sigma).
Analysis of GAL₂ receptor IR was performed under light microscopy (Nikon Optiphot-2) in the neuro-anatomical area of interest. Four sections of two animals per day were examined and captured by a video camera (Olimpus UC30) linked to a PC computer, and GAL₂ receptor IR was quantified by optical density using the computer software ImageJ (National Institutes of Health).

Millón C., Flores-Burgess A., Narváez M., Borroto-Escuela D.O., Santín L., Parrado C., Narváez J.A., Fuxe K, & Díaz-Cabiale Z. (2015). A Role for Galanin N-Terminal Fragment (1–15) in Anxiety- and Depression-Related Behaviors in Rats. International Journal of Neuropsychopharmacology, 18(3), pyu064.

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Immunohistochemical Analysis of Bone Markers

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Sections (Results μM thick) were deparaffinized in three changes of xylene and rehydrated in a graded series of ethanol solutions (ending with distilled water). For antigen retrieval, slides were placed in 0.01 M citrate‐buffer, pH 6.0, and heated in a steamer for 30 minutes. Endogenous peroxidases were quenched by incubating with 3% hydrogen peroxide for 20 minutes at room temperature. Sections were incubated overnight at 4°C with a 1:50 dilution of primary antibody: anti‐collagen Col1 (Santa‐Cruz Biotechnology), anti‐BSP (Santa‐Cruz Biotechnology), anti‐OC (Santa‐Cruz Biotechnology), or anti‐NFATC1 (Santa‐Cruz Biotechnology). Subsequently, sections were incubated for 30 minutes with a biotinylated secondary antibody (LSAB; Dako Cytomation, Glostrup, Denmark), washed in PBS, and incubated for 30 minutes with a streptavidin‐peroxidase conjugate (LSAB, Dako Cytomation). The reaction was developed for 5 minutes using 3,30‐diaminobenzidine tetrahydrochloride (Sigma‐Aldrich). Slides were briefly counterstained in hematoxylin, dehydrated, and cover slipped. Negative and positive controls were run simultaneously. Positive controls comprised mammary tissue. The slides were captured using a microscope slide scanner (3D‐HISTECH Ltd.).

Kim D., Ko Y., Park M., Kim B., Sohn H, & Lim W. (2019). Regulation of Osteosclerosis by Inoculated Cd133+ PC3 Cells in Bone‐marrow Microenvironmental Niches. JBMR Plus, 3(7), e10189.

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Immunohistochemical Analysis of AT2R Expression

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Lung tissues fixed with 10% buffered formalin were sectioned at 4 µm and stained with hematoxylin and eosin (H&E) for histologic examination. To analyze AT2R expression in both LLC and H358 tumor grafts, sections were deparaffinized and heat-induced epitope unmasking was performed in the citrate buffer followed by incubation with 3% H₂O₂/methanol for 3 minutes to block endogenous peroxidase activity. Sections were incubated with polyclonal anti-AT2R antibodies (1:200 dilution, for 18 hours at 4°C, Santa Cruz Biotechnology, Inc., Dallas, TX). After the incubation with primary antibodies, sections were incubated with a biotin-conjugated anti-rabbit IgG antibody (Vector Laboratories, Burlingame, CA) at a 1:100 dilution for 1 hour at 37°C, followed by a reaction with the avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 40 minutes at 37°C. Reactions were developed with 3, 30-diaminobenzidine tetrahydrochloride (Sigma) and the sections were counterstained lightly with Mayer hematoxylin.

Ishiguro S., Alhakamy N.A., Uppalapati D., Delzeit J., Berkland C.J, & Tamura M. (2016). Combined local pulmonary and systemic delivery of AT2R gene by modified TAT peptide nanoparticles attenuates both murine and human lung carcinoma xenografts in mice. Journal of pharmaceutical sciences, 106(1), 385-394.

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Immunohistochemical Analysis of Titin in Mouse EDL

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Mouse EDL muscles were fixed in 4% paraformaldehyde for 24 hr, paraffin embedded, and cut in 5 mm sections with a vibratome. Each section was incubated with 3% H 2 O 2 in methanol for 30 min to quench endogenous peroxidase activity. Tissue sections were dehydrated with graded alcohols and exposed to 10 mM sodium citrate for antigen retrieval. The sections were blocked with PBS containing 5% normal goat serum and 0.3% Triton X-100 for 1 hr at room temperature and then incubated with anti-TTN3 (1:100) antibody diluted in PBS containing 1% BSA and 0.3% Triton X-100 overnight at 4 C. The sections were subsequently washed three times, incubated for 1 hr with HRP-conjugated secondary antibody, incubated with 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma) after wash, and immediately washed under tap water after the color development. Slides were stained with H&E.

Hong G.S., Lee B., Wee J., Chun H., Kim H., Jung J., Cha J.Y., Riew T.R., Kim G.H., Kim I.B, & Oh U. (2016). Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function. Neuron, 91(1).

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Histochemical Localization of Hyaluronan

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HA localization was determined using the biotinylated HA-binding protein by modification of the technique described previously. 31 Briefly, after blocking of endogenous peroxidase activity, the sections were incubated with 2 mg/mL biotinylated HA-binding protein (Hokudo, Sapporo, Japan) in phosphate-buffered saline containing 10% fetal bovine serum for 1 hour. After washing in phosphate-buffered saline, the slides were incubated with streptavidin horseradish peroxidase (BD Pharmingen) for 1 hour, washed in phosphate-buffered saline, and incubated in a solution of 3,3 0 -diaminobenzidine tetrahydrochloride (Sigma-Aldrich). They were counterstained with hematoxylin. Control slides were pre-incubated with 200 tenfold reduction units/mL Streptomyces hyaluronidase Table 1 Primer Sequences Used for Real-Time Quantitative PCR Analysis

Shimoda M., Yoshida H., Mizuno S., Hirozane T., Horiuchi K., Yoshino Y., Hara H., Kanai Y., Inoue S., Ishijima M, & Okada Y. (2017). Hyaluronan-Binding Protein Involved in Hyaluronan Depolymerization Controls Endochondral Ossification through Hyaluronan Metabolism. The American journal of pathology, 187(5).

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