Harvested subcutaneous tumors were formalin-fixed and paraffin-embedded. Sectioned tissues were then deparaffinized and soaked in 0.3% H2O2 in methanol at RT for 10 minutes to extinguish endogenous peroxidase activity. Antigen retrieval was performed by heating specimens in a sodium citrate buffer solution using a microwave. After cooling, sections were incubated in Peroxidase Blocking Reagent (Dako, Santa Clara, USA) for 10 minutes at RT. Sectioned tissues were incubated with primary antibody against CD8 (clone 4SM15, 1:100 dilution, eBioscience, San Diego, USA) or FoxP3 (clone FJK-16s, 1:100 dilution, eBioscience) or aSMA (A5228, 1:1,000 dilution, Sigma-Aldrich) or CD4 (clone 4SM95, 1:100 dilution, eBioscience) or TGF-β (ab215715, 1:100 dilution, Abcam, Cambridge, UK) for 60 minutes at RT. Following three 5-minute washes with PBS, sections were incubated with secondary antibody for 30 minutes at RT. After washing, the enzyme substrate 3,30-diaminobenzidine (Dako, Santa Clara, USA) was used for visualization, and sections were counterstained with Meyer’s hematoxylin. Evaluation of sections were performed using ImageJ software. The number of cells expressing CD8, FoxP3, CD4 were counted in four randomly selected high-magnification fields. The scores of αSMA and TGF-β were evaluated using an “area index,” calculated in low magnification fields.
3 30 diaminobenzidine
Manufactured by Agilent Technologies
Sourced in Denmark, United States
3,30-diaminobenzidine is a chemical reagent commonly used in various laboratory applications. It serves as a chromogenic substrate for the detection and visualization of enzymatic activity, particularly in immunohistochemistry and cytochemistry techniques. The core function of 3,30-diaminobenzidine is to produce a colored precipitate upon enzymatic catalysis, allowing for the identification and localization of target molecules or cells of interest.
Automatically generated - may contain errors
Lab products found in correlation
Photomicroscope, by Olympus (2 mentions)
Streptavidin peroxidase complex, by Vector Laboratories (2 mentions)
Ab215715, by Abcam (1 mentions)
Peroxidase blocking reagent, by Agilent Technologies (1 mentions)
Clone 4sm15, by Thermo Fisher Scientific (1 mentions)
Clone 4sm95, by Thermo Fisher Scientific (1 mentions)
Clone fjk 16s, by Thermo Fisher Scientific (1 mentions)
A5228, by Merck Group (1 mentions)
Citrate buffer, by Vector Laboratories (1 mentions)
Bz x800 microscope, by Keyence (1 mentions)
Wst 8 kit, by Dojindo Laboratories (1 mentions)
Ab6640, by Abcam (1 mentions)
Picrosirius red stain kit, by Polysciences (1 mentions)
Gtx100034, by GeneTex (1 mentions)
Nbp1 85047, by Novus Biologicals (1 mentions)
Hrp linked secondary antibody, by Agilent Technologies (1 mentions)
Il 1β, by Santa Cruz Biotechnology (1 mentions)
Timp3, by Abcam (1 mentions)
Interleukin 10 il 10, by Abcam (1 mentions)
Mcp 1, by Abcam (1 mentions)
13 protocols using 3 30 diaminobenzidine
1
Immunohistochemical Analysis of Tumor Microenvironment
2
Immunohistochemical Analysis of HOPX in Cardiac Tissue
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Immunohistochemistry (IHC) was performed using an ABC immunoperoxidase staining kit (Vector Laboratories, Burlingame, CA, USA). In brief, cardiac tissue sections were deparaffinized using xylene. Antigen retrieval was performed in a microwave for 20 min with 1X citrate buffer (Vector Laboratories). Endogenous peroxidase activity was prevented by treating the tissue sections with 3% hydrogen peroxide. Tissue sections were blocked with the 2.5% normal horse serum (Vector Laboratories) at RT for 1 h. Tissue sections were incubated with the HOPX primary antibody (Thermo Fisher Scientific) at 1:500 dilution for 1 h at RT. Slides were washed with the 1X TBST and incubated with biotinylated anti-rabbit antibody for 30 min and then developed with 3, 3, 0-diaminobenzidine (Dako, Santa Barbara, CA, USA). Furthermore, tissue sections were counterstained with hematoxylin. Images of the stained slides were captured using a BZ-X800 Keyence microscope (Keyence).
3
Comprehensive Analysis of Cell Proliferation, Motility, and Angiogenesis
4
Histological and Immunohistochemical Evaluation of Liver and Intestinal Tissues
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The formalin-fixed liver sections were stained with H & E, Sirius red, and Masson’s trichrome to detect any liver injury and fibrosis. Further, intestine sections were stained with H & E. The liver HE-stained sections were scored using the NAFLD activity score (NAS) system (Brunt et al., 2011 (link)) by our co-author H.-S.L.—an experienced pathologist—and the degree of fibrosis was evaluated by Sirius red stain (Picrosirius Red Stain Kit, Polysciences, Inc., Warrington, PA, United States) and Masson’s trichrome staining (Muto pure chemical co., Ltd.). NIH ImageJ was used to estimate the length and width of gut villi and crypts.
Paraffin-embedded liver sections were stained for F4/80 (ab6640, abcam®) and stained for alpha-smooth actin (a-SMA) (GTX100034, GeneTex). Paraffin-embedded intestine sections were stained for ZO-1 (NBP1-85047, Novus). Standard immunohistochemical (IHC) staining procedures were employed; liver and intestine sections were incubated with a specific primary antibody, as described earlier, followed by incubation with an HRP-linked secondary antibody (Dako, Glostrup, and Denmark) and 3,30-diaminobenzidine (Dako, Glostrup, and Denmark) and were scanned using the NanoZoomer Digital Pathology system.
Paraffin-embedded liver sections were stained for F4/80 (ab6640, abcam®) and stained for alpha-smooth actin (a-SMA) (GTX100034, GeneTex). Paraffin-embedded intestine sections were stained for ZO-1 (NBP1-85047, Novus). Standard immunohistochemical (IHC) staining procedures were employed; liver and intestine sections were incubated with a specific primary antibody, as described earlier, followed by incubation with an HRP-linked secondary antibody (Dako, Glostrup, and Denmark) and 3,30-diaminobenzidine (Dako, Glostrup, and Denmark) and were scanned using the NanoZoomer Digital Pathology system.
5
Immunohistochemical Protein Expression Analysis
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IHC was performed to evaluate the protein expression of a candidate gene with a specific antibody against the protein as reported previously.[11 (link)
] In brief, paraffin sections were deparaffinized with xylene and rehydrated, followed by antigen retrieval. The sections were then treated with 3% hydrogen peroxide to quench the endogenous peroxidase activity, followed by incubation with a primary antibody overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, chromogenic immunolocalization was conducted using 0.05% 3,30‐diaminobenzidine (Dako, Glostrup, Denmark). All sections were counterstained with hematoxylin. IHC evaluation was performed independently by three pathologists at SYSUCC. An IHC staining score was calculated as the product of staining intensity multiplied by the percentage of stained cells (TableS3 , Supporting Information). The staining intensity was evaluated on a scale of 0 to 3: 0 for no, weak, intermediate, and strong staining, respectively.
] In brief, paraffin sections were deparaffinized with xylene and rehydrated, followed by antigen retrieval. The sections were then treated with 3% hydrogen peroxide to quench the endogenous peroxidase activity, followed by incubation with a primary antibody overnight at 4 °C. After incubation with secondary antibodies for 1 h at room temperature, chromogenic immunolocalization was conducted using 0.05% 3,30‐diaminobenzidine (Dako, Glostrup, Denmark). All sections were counterstained with hematoxylin. IHC evaluation was performed independently by three pathologists at SYSUCC. An IHC staining score was calculated as the product of staining intensity multiplied by the percentage of stained cells (Table
6
Immunohistochemical Analysis of Inflammatory Markers
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Sections were deparaffinized and rehydrated using a graded ethanol series and incubated overnight at 4 °C with antibodies to interleukin (IL)-1β (Santa Cruz Biotechnology, Santa Cruz, CA, USA), matrix metalloproteinase-3 (MMP-3; Abcam, Cambridge, UK), tissue inhibitor of metalloproteinase-3 (TIMP-3) (Abcam), Interleukin-10 (IL-10) (Abcam), CCR2 (Novus Biologicals, Littleton, CO, USA), GABA (Novus Biologicals), and monocyte chemoattractant protein (MCP)-1 (Abcam). The slides were then treated with secondary antibodies and biotinylated anti-mouse IgG for 20 min conjugated to streptavidin peroxidase complex (Vector Laboratories, Burlingame, CA, USA) for 1 h, and then treated with 3,30-diaminobenzidine (Dako, Glostrup, Denmark). The slides were counterstained with Mayer’s hematoxylin and photographed under a photomicroscope (Olympus, Tokyo, Japan). Immunohistochemistry evaluations were performed independently by two experienced researchers who were blinded to the study groups. The positive cell percentage was analyzed with HDAB (hematoxylin and DAB) by selecting color deconvolution in the plugin item in the image J program (NIH, MD, USA).
7
Sural Nerve Biopsy Analysis Protocol
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Sural nerve biopsy was performed after obtaining informed consent, as previously described. Light and electron microscopy preparations, as well as teased fiber analysis, were performed according to standard methods [20 (link)]. Immunohistochemistry was performed in selected cases (see below). Sections were deparaffinized in xylene, rehydrated through decreasing concentrations of ethyl alcohol and then rinsed in distilled water. Antigen retrieval was performed in a 90 °C solution of 0.001 M EDTA buffer (pH 8.0) for 20 min. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in distilled water. Slides were incubated with antibodies recognizing CD3 (clone F7.2.38, 1:50, Dako), CD20 (clone L26, 1:50, Dako) and CD68 (clone KP1, 1:1000, Dako) for 1 h at room temperature. Bound antibodies were detected using Envision FLEX/HRP and 3–30-diaminobenzidine as chromogen (Dako). Slides were counterstained with haematoxylin (Sigma-Aldrich), dehydrated and mounted.
8
Immunohistochemical Quantification of MPO
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Immunohistochemical staining was performed on 4-µm paraffinized sections. The samples were dewaxed, dehydrated, washed in PBS, incubated with 3% H2O2 for 20 min to quench endogenous peroxidase activity and incubated with normal goat serum (1:20) for 30 min. Afterward, the samples were treated with anti-MPO antibody (rabbit polyclonal, 1:500; Servicebio, China) at 4 °C overnight. The sections were then incubated with a horseradish peroxidase-conjugated secondary antibody (anti-rabbit IgG; Servicebio, China) for 2 h at room temperature. After being washed three times with PBS, the sections were stained with 3,30-diaminobenzidine (DAKO, China) and subsequently stained with hematoxylin. Then, the number of positive cells was evaluated under a light microscope. Ten areas were randomly selected. The sections were scored according to the number of MPO-positive cells per high power field (HP) in a blinded manner.
9
Immunostaining for Cell Proliferation and Apoptosis
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To study cell proliferation and apoptotic cell death, proliferating cell nuclear antigen (PCNA) and cleaved caspase 3 were stained with indirect immunoperoxidase techniques. 20, 21 Therefore, deparaffinized sections were incubated with 3% H 2 O 2 and 2% goat normal serum to block endogenous peroxidases and unspecific binding sites. A monoclonal rat anti-pan PCNA antibody (PC10, 1:50; DakoCytomation, Hamburg, Germany) and a polyclonal rabbit anti-rat cleaved caspase 3 antibody (Asp175, 1:50; Cell Signaling Technology, Frankfurt, Germany) were used as primary antibodies. The cleaved caspase 3 antibody detects endogenous levels of the short fragment (17/19kD) of activated caspase 3 but not full-length caspase 3. Biotinylated goat anti-rat and goat anti-rabbit IgG antibodies were used as secondary antibodies for streptavidin-biotin complex peroxidase staining (1:200, Labeled Streptavidin-Biotin 2 System horseradish peroxidase; DakoCytomation); 3,3 0 -diaminobenzidine (DakoCytomation) was used as a chromogen. Sections were counterstained with hemalaun and examined by light microscopy. Cell proliferation and apoptotic cell death are given as numbers of positive cells per high-power field.
10
Immunohistochemical Analysis of Pituitary Adenoma
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Formalin-fixed paraffin-embedded pituitary adenoma blocks were obtained from the Biobank (University Hospitals Leuven). Antigen retrieval was performed on 5 mm sections with EnVision FLEX Target Retrieval Solution (Dako, Glostrup, Denmark). Endogenous peroxidase activity was blocked with EnVision Peroxidase-Blocking Reagent, and sections were incubated with primary antibodies directed against human E-cadherin (readyto-use; Dako), vimentin (VIM; 1:500; Dako), NOTCH2 (1:200; Abcam, Cambridge, UK), and interleukin 6 (IL6; 1:200; Abcam). Subsequently, samples were processed using EnVision Dual Link (Dako). The complex formed was visualized with 3,3 0 -diaminobenzidine (Dako), and sections were counterstained with hematoxylin (Dako).
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