Immunohistochemical staining of the CRC tissues was performed using a tissue microarray (TMA) block of 343 patient tissues. The tissue core punched by a 2-mm puncher (Unitech Korea Co., Ltd.) was embedded in a recipient paraffin block (Unitech Korea Co., Ltd.). The TMA blocks were cut into 4-µm sections, dewaxed in xylene and rehydrated through a gradient concentration of ethanol (100, 95, 90, 80 and 70%) for 5 min per concentration. Subsequently, the blocks were incubated in 0.01 M citrate buffer (pH 6.0) at 95°C for 30 min in a microwave. Endogenous peroxidase activity was inactivated using 0.3% H2O2 for 1 h at room temperature. The sections were subsequently incubated with antibodies against TET2 (1:100; cat. no. ab245287; Abcam) and AMPK (1:100; cat. no. GTX52341; GeneTex, Inc.) overnight at 4°C, followed by incubation with an anti-rabbit EnVision secondary antibody (cat. no. K4002; Dako; Agilent Technologies, Inc.) for 1 h at 37°C. For visualization, the sections were treated with µl 3,3′-diaminobenzidine solution (Dako; Agilent Technologies, Inc.) and counterstained with Harris' hematoxylin (EMD Millipore). The sections were mounted using Canada Balsam (Sigma-Aldrich; Merck KGaA).
3 3 diaminobenzidine solution
Manufactured by Agilent Technologies
Sourced in Denmark, Germany
3,3'-diaminobenzidine solution is a chemical reagent commonly used in various laboratory applications, particularly in histochemical and immunohistochemical staining procedures. It serves as a chromogenic substrate that undergoes an enzymatic reaction, resulting in the production of a colored insoluble precipitate. This solution is often utilized to detect the presence and localization of specific proteins or enzymes in biological samples.
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Lab products found in correlation
Harris hematoxylin, by Merck Group (1 mentions)
Canada balsam, by Merck Group (1 mentions)
Mmp 13, by Bioss Antibodies (1 mentions)
Il 1β, by Merck Group (1 mentions)
Horseradish peroxidase, by Agilent Technologies (1 mentions)
Optical microscope, by Olympus (1 mentions)
Ab79823, by Abcam (1 mentions)
Dako protein block serum free, by Agilent Technologies (1 mentions)
Hematoxylin, by Merck Group (1 mentions)
Lab tek chamber slide, by Thermo Fisher Scientific (1 mentions)
Anti cdc2 antibody, by Cell Signaling Technology (1 mentions)
Ckx41, by Olympus (1 mentions)
Protein block solution, by Agilent Technologies (1 mentions)
Peroxidase blocking solution, by Agilent Technologies (1 mentions)
Normal rabbit igg, by Santa Cruz Biotechnology (1 mentions)
Digital sight ds u2 camera, by Nikon (1 mentions)
Blocking solution, by Agilent Technologies (1 mentions)
Target retrieval solution, by Agilent Technologies (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Anti collageniiprimary antibody, by Abcam (1 mentions)
11 protocols using 3 3 diaminobenzidine solution
1
Immunohistochemical Staining of CRC Tissues
2
Immunoglobulin G Deposition in Glomeruli
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To detect immunoglobulin IgG deposition in glomeruli, kidney sections were stained with a biotinylated goat anti-murine IgG (Jackson ImmunoResearch Labs). Sections were then washed and incubated with a streptavidin-linked horseradish peroxidase (Agilent). Bound antibodies were visualized following incubation with 3,3’-diaminobenzidine solution (0.05% with 0.015% H2O2 in PBS; Agilent). Sections were counterstained with Mayer’s hematoxylin.
3
Immunohistochemical Analysis of Osteoarthritis Markers
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For immunohistochemistry staining, the tibial articular sections were rehydrated, and endogenous peroxidase in the tissue was blocked with 3% hydrogen peroxide. Sections were then blocked with 10% FBS for 1 h and incubated with primary antibodies against type II collagen (1:2000; mouse monoclonal antibody; Chemi-Con, Temecula, CA, USA), IL1-β (1:500; rabbit polyclonal antibody; Merck, KGaA, Darmstadt, Germany), ADAMTS5 (1:1000; rabbit polyclonal antibody; Novus Biologicals, LLC, Centennial, CO, USA), and MMP13 (1:200; rabbit polyclonal antibody; Bioss, MA, USA) at 4 °C overnight. Then, the samples were incubated for 30 min with biotin-labeled goat anti-mouse/rabbit immunoglobulin and horseradish peroxidase-conjugated streptavidin (BioCare Medical, Pacheco, CA, USA) secondary antibodies. Staining with a 3,3′-diaminobenzidine solution (Dako) containing 0.01% hydrogen peroxide resulted in a brown color. Finally, sections were counterstained with hematoxylin and observed under a microscope. The relative density of immunostaining (density/area; mean ± SEM area 25.44 ± 2.77 mm2) was measured using the Image-Pro Plus software, version 5.0.
4
Immunohistochemical Analysis of GATA4 and MUC2
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Formalin‐fixed paraffin‐embedded tissue slices were processed for deparaffination and rehydration. Slices were then processed with primary antibodies against human GATA4 (1:100; Santa Cruz Biotechnology), human MUC2 (1:15,000; Abcam), and mouse Gata4 (1:100; Abmart, T56814). Following treatment with secondary antibodies conjugated to horseradish peroxidase (Dako, Glostrup, Denmark) for 30 min, the slices were developed with 3,3’-diaminobenzidine solution (Dako). Results were scored independently by at least two professional pathologists. The immunoreactivity score was computed by multiplying the intensity of the staining by the proportion of positive cells. The intensity of the staining was measured as indicated below: 0, negative; 1, weak; 2, moderate; 3, strong. The positive rate of staining was scored as follows: 0, <10%; 1, 10% to 25%; 2, 26% to 50%; 3, 51% to 75%; 4, >75%.
5
Immunohistochemical Analysis of HMGB1
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The paraffin-embedded tissues were cleansed of paraffin and rehydrated using graded alcohols. Endogenous peroxidase activity was blocked with 3% H2O2 at 10 minutes and was washed using PBS. Antigen retrieval was performed by boiling the slides in 10 mM citrate buffer (pH 6.0) for 5 minutes. Anti-high mobility group box 1 (HMGB1) (ab79823; Abcam) was used as the primary antibody and incubated at 4°C overnight. Negative controls were prepared using PBS in place of the primary antibodies. After that, the slides were incubated with an anti-rabbit horseradish peroxidase-conjugated secondary antibody at room temperature for 2 hours. Sections were developed with 3, 3′-diaminobenzidine solution (DAKO, Brüsseler, Germany) at room temperature for 1 minute. The slides were finally observed under an optical microscope (Olympus, Tokyo, Japan) at a magnification of × 400.
6
Immunohistochemistry for SUCNR1 Receptor
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Paraffin was removed from the tissues after warming slides to 60°C with xylene. Tissue was rehydrated stepwise in decreasing ethanol concentrations and epitopes were retrieved in heated citrate buffer (10 mM sodium citrate, 1 mM citric acid, pH 6). Tris-buffered saline with 0.1% tween 20 was used for wash steps. Endogenous peroxidases were inhibited with 3% H2O2 followed by DAKO protein block serum-free (Dako, Glostrup, Denmark). Slides were incubated with rabbit anti-SUCNR1 antibody (ab140795 Abcam, Cambridge, UK) in blocking solution in a humidified chamber for 1 h at 37°C. Peroxidase-labeled polymer conjugated with secondary goat anti-rabbit antibody (Dako EnVision) was applied. Visualization was based on the peroxidase reaction with 3,3-diaminobenzidine solution (Dako). Tissue was counterstained with hematoxylin. Dehydration was performed by stepwise immersion in increasing ethanol concentrations followed by xylene before mounting.
7
Immunocytochemical Analysis of Cdk1 and Cdc2 in Ovarian Cancer Cell Lines
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Immunocytochemistry was performed on methanol-fixed OVCA-429, OVCAR-3 and SK-OV-3 cell lines using anti-Cdk1 antibody (Thermo Scientific, Rockford, IL) and anti-Cdc2 antibody (Cell Signaling Technology, Danvers, MA). Briefly, after seeding 5 × 105 cells per well in Lab-Tek Chamber slide (Nunc, Rochester, NY), cell lines were treated with ice cold methanol for fixation. Then the endogenous peroxidase activity was quenched by 3% hydrogen peroxide solution for 10 min. Non-specific binding was prevented by incubation with 5% bovine serum albumin and 0.01% Triton X-100 with 1 × Phosphate buffer saline for 15 min. After that, the sections were incubated with anti-Cdk1 antibody (1:300 dilutions) and anti-Cdc2 antibody (1:630 dilutions) for overnight at 4°C. Antibody binding was detected using horseradish peroxidase-conjugated secondary antibody at 37°C for 30 min. Then sections were visualized by 3,3′-diaminobenzidine solution (DAKO, Seoul, Republic of Korea), counterstained lightly with hematoxylin (Sigma-aldrich) dehydrated with ethanol and observed using inverted microscopy (model CKX41, Olympus).
8
Immunohistochemical Analysis of NLRX1 in Murine Lungs
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The left lobes of sacrificed mice lungs were fixed in 4% formalin for 3 days and embedded in paraffin. Lung tissues were cut into 5-µm sections and stained using hematoxylin and eosin (H&E) to analyze airway inflammation. For immunohistochemical studies, sections were deparaffinized by washing twice with xylene for 10 min followed by treatment with 100% (5 min × 2), 95% (5 min), and 70% (5 min) ethanol. The tissues were then heated with retrieval buffer (DAKO, A/S, Glostrup, Denmark) for 20 min in a steamer and allowed to cool to room temperature (RT; 18–22 °C) over 20 min. After washing the sections, they were placed in peroxidase blocking solution (DAKO) for 5 min, and then in protein block solution (DAKO) for 1 h. The NLRX1 antibody (Proteintech, Rosemont, IL) or normal rabbit IgG (Santa Cruz Biotechnology, Inc, Dallas, TX) were prepared as 1:500 dilutions and applied to tissue sections, followed by incubation at 4 °C overnight. The color was developed with 3,3′-diaminobenzidine solution (DAKO), and the reaction was stopped using deionized water; the immunostained tissue sections were mounted on slides with an aqueous-base mounting medium.
9
Immunohistochemical Analysis of Cystatin C
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Sections were deparaffinized and rehydrated in xylene and ethanol. They were then immunostained using the EnVision Detection System Peroxidase/DAB, Rabbit/Mouse kit (Dako, K4065). Incubations with primary antibody (anti-Cystatin C, Sigma, HPA013143, used at 1:100 dilution) were performed at room temperature for 30 min. After incubation with a primary antibody, sections were incubated with EnVision FLEX/HRP. Sections were washed between steps with Tris-buffered NaCl solution with Tween 20, pH 7.6 (Dako, S3306). All sections were incubated with 3,3′-diaminobenzidine solution (Dako, K4065) for 10 min. Sections were counterstained with haematoxylin for 5 min followed by washing with tap water for 10 min. Finally, sections were dehydrated with 100% ethanol and xylol followed by coverslipping with mounting medium (Pertex, Histolab). Images were acquired with a Nikon Eclipse 50i microscope equipped with a Nikon DS-Fil digital camera and a Nikon Digital Sight DS-U2 camera controller. Image panels were constructed using the GNU Image Manipulation Program (GIMP 2.8.10).
10
Immunohistochemical analysis of IL-6 and p-IKKa/b
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Formalin-fixed, paraffin-embedded tissue blocks were sliced into sections (4 mm thick). The sections were deparaffinized in xylene and dehydrated with 100% ethanol. Antigen unmasking was performed by boiling the sections in Target Retrieval Solution (pH 9.0; Dako, Glostrup, Denmark). After being placed in 3% H 2 O 2 and blocked with Blocking Solution (Dako), the sections were incubated with the primary antibodies against IL-6 and p-IKKa/b at 1:40 for 1 hour at room temperature. After washing with Tris-buffered saline containing 0.1% Tween-20, the slides were stained using the Envision system (Dako), followed by the application of 3,3-diaminobenzidine solution (Dako) until the wanted stain intensity developed, and then were counterstained with Mayer's hematoxylin.
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