Mouse tumors and lungs were dissected and fixed in 4% paraformaldehyde and embedded in paraffin. Tissue sections at 4 µm were immunostained following standard procedures.25 (link) We used Ki-67 (ab15580; Abcam, Cambridge, UK), CD34 (GTX61737; GeneTex, Irvine, CA, USA), MMP2 (GTX104577; GeneTex), MMP9 (GTX61537; GeneTex), CD206 (ab64693; Abcam), and inducible nitric oxide synthase (iNOS, ab15323; Abcam) as primary antibodies. Goat anti-rabbit IgG conjugated with horseradish peroxidase (Boster, Wuhan, China) was applied as secondary antibody. Positive reactions were colorized with 3-amino-9-ethylcarbazole (Zymed Laboratories, South San Francisco, CA, USA) and counterstained with hematoxylin. The slides were photographed with a Motic light microscope (Motic, Xiamen, China).
3 amino 9 ethylcarbazole
Manufactured by Zymo Research
Sourced in United States
3-amino-9-ethylcarbazole is a chemical compound used as a chromogenic substrate in various laboratory techniques, such as immunohistochemistry and enzyme-linked immunosorbent assays (ELISA). It is a sensitive and stable substrate that produces a brown-colored precipitate upon oxidation, allowing for the visualization and detection of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Avidin biotin complex reagent, by Agilent Technologies (2 mentions)
Goat anti rabbit igg conjugated with horseradish peroxidase, by Boster Bio (1 mentions)
Cd206, by Abcam (1 mentions)
Ab15323, by Abcam (1 mentions)
Envision system, by Agilent Technologies (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Type 1 collagen, by Abcam (1 mentions)
Interleukin 1β il 1β, by Cell Signaling Technology (1 mentions)
Matrix metalloproteinase 9 mmp 9, by Abcam (1 mentions)
Anti nitrotyrosine, by Abcam (1 mentions)
Histoquest analysis software, by TissueGnostics (1 mentions)
Rat elisa kit, by R&D Systems (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Jackson ImmunoResearch (1 mentions)
Anti vwf antibody, by Agilent Technologies (1 mentions)
Universal immpress kit, by Vector Laboratories (1 mentions)
Vectastain elite abc peroxidase kit, by Vector Laboratories (1 mentions)
Vectamount aq aqueous mounting medium, by Vector Laboratories (1 mentions)
7 protocols using 3 amino 9 ethylcarbazole
1
Immunohistochemical Analysis of Mouse Tumor and Lung Samples
2
Immunohistochemical Detection of PPAR-α in Ampullary Cancer
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Formalin-fixed paraffin-embedded sections of ampullary cancer were obtained from the Human Biobank of National Cheng Kung University Hospital. Slides were deparaffinized, rehydrated, and underwent heat-retrieval in a pressure boiler. Expression of the PPAR-α protein was detected by a polyclonal rabbit anti-PPAR-α antibody (GeneTex, Irvine, CA, USA) and a goat anti-rabbit secondary antibody conjugated with a peroxidase-labeled polymer (EnVisionTM System, Dako, Denmark). Color was developed with 3-amino-9-ethyl carbazole (Zymed, Waltham, MA, USA), and nuclei were counterstained with Mayer's hematoxylin. Slides were evaluated as low or high expression of PPAR-α.
3
Nestin Expression in Ampullary Adenocarcinoma
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Tissue of ampullary adenocarcinoma and the normal duodenum were fixed in 4% formalin and embedded in paraffin. Serial sections were cut from each sample. All samples were acquired from the Human Biobank at the Research Center of Clinical Medicine of NCKUH after obtaining appropriate informed consent. The study was approved by the Institutional Review Board (NCKUH IRB no: A-ER-100-395).
IHC staining was performed using a monoclonal mouse anti-human nestin antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The sections were incubated using an avidin-biotin complex reagent (Dako, Carpinteria, CA, USA) and final color was developed with 3-amino-9-ethyl carbazole (Zymed Laboratories, Inc., San Francisco, CA, USA). The sections were counterstained with hematoxylin. The internal positive controls consisted of endothelial cells, and primary or secondary antibodies were omitted to serve as negative controls. The immunoreactivity of the nestin protein was assessed using the semi-quantitative method and scaled according to the immunoreactive score (IRS) established by Remmele and Schicketanz (21 (link)). The IRS points ranged from 0 to 12 and were divided into 4 classifications: negative, weak, mild and strong. One researcher assessed all lesions (H.-P.H).
IHC staining was performed using a monoclonal mouse anti-human nestin antibody (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The sections were incubated using an avidin-biotin complex reagent (Dako, Carpinteria, CA, USA) and final color was developed with 3-amino-9-ethyl carbazole (Zymed Laboratories, Inc., San Francisco, CA, USA). The sections were counterstained with hematoxylin. The internal positive controls consisted of endothelial cells, and primary or secondary antibodies were omitted to serve as negative controls. The immunoreactivity of the nestin protein was assessed using the semi-quantitative method and scaled according to the immunoreactive score (IRS) established by Remmele and Schicketanz (21 (link)). The IRS points ranged from 0 to 12 and were divided into 4 classifications: negative, weak, mild and strong. One researcher assessed all lesions (H.-P.H).
4
Histological Evaluation of Tendinopathy
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The mice were killed, and theircollagenase I-treated tendon tissue were fixed with 4% paraformaldehyde, and stained with hematoxylin & eosin to evaluate the change of collagen alignment. Based on our previous study described in detail [22 (link)], a modified semiquantitative 4-point scoring method for each factor was used. Tendinopathy severity, based on the sum of the scores, was graded as 0–3 (0, ≤ 2, 3–4, ≥5 points). After scoring, the tendon tissue were subjected to undergo immunohistochemistry and TUNEL assay. The tendon tissue from normal and overiectomy rats (under static and stretch conditions) were immunohistochemically analyzed. The processed tissuewere snap-frozen and embedded in paraffin. The sections were deparaffinized in xylene, dehydrated in alcohol, treated with proteinase K, washed with H2O2 in PBS, and stained with antibodies against ER-β (Abcam), collagen type I (Abcam), cleaved caspase 3 (Cell Signaling Technology), interleukin-1β (IL)-1β (Cell Signaling), and matrix metalloproteinase-9 (MMP)-9 (Abcam), in combination with the chromogen 3-amino-9-ethylcarbazole (Zymed). The signal intensity was further quantitated using Image J 1.42q (National Institutes of Health) in three randomly chosen fields.
5
Synovial Inflammation Biomarker Quantification
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After de-paraffinization and re-hydration, 5 µm synovial sections were stained with anti-nitrotyrosine (Abcam, Cambridge, UK) or anti-vWF antibody (DAKO, Agilent, Santa Clara, CA, USA), followed by horseradish-peroxidase-labeled secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA) and substrate chromogen 3-amino-9-ethylcarbazole (Zymed Laboratories, South San Francisco, CA, USA), and expression intensities were quantified using the HistoQuest analysis software (TissueGnostics, Tarzana, CA, USA) as described previously [15 (link)]. The removed joints were skinned and further homogenized in 1 mL of radioimmunoprecipitation assay (RIPA) lysis buffer containing a protease inhibitor cocktail (Pierce Manufacturing, Appleton, WI, USA). Then, the concentrations of interleukin (IL)-1β, tumor necrosis factor (TNF), and vascular endothelial growth factor (VEGF) were measured using rat ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol, as described previously [17 (link)].
6
Immunohistochemistry of TGR5 in Ampullary Adenocarcinoma
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Samples of ampullary adenocarcinoma and the surrounding duodenum were fixed in 4% formalin and embedded in paraffin. IHC staining was performed using a monoclonal mouse anti-human TGR5 antibody (Abcam Biotechnology). The sections were incubated using an avidin-biotin complex reagent (Dako, Carpinteria, CA, USA), incubated with 3-amino-9-ethyl carbazole (Zymed Laboratories, South San Francisco, CA, USA) to develop the final color, and counterstained with hematoxylin. The immunoreactivity of the TGR5 protein was assessed using a semi-quantitative method and according to the Remmele and Stegner immunoreactive scoring (IRS) system (26 (link)). The IRS scores ranged from 0 to 12 and immunoreactivity was characterized as negative, weak, mild and strong. One researcher assessed the lesions (H.P. Hsu).
7
Immunohistochemical Analysis of Immune Cells
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Tissue sections were deparaffinized in xylene and rehydrated with decreasing concentrations of alcohol. Subsequently, endogenous peroxide was blocked with hydrogen peroxide, and antigen retrieval was achieved by treating sections with 0.01 m sodium citrate, pH 6.0 for 1 min in a pressure cooker. After blocking with universal blocking solution (ZYMED Laboratories, San Francisco, CA, USA), tissue sections were stained with the designated primary antibodies. A Vectastain Elite ABC Peroxidase kit or Universal ImmPRESS kit (Vector Laboratories, Burlingame, CA, USA) were used for secondary antibodies, and visualization was performed using 3-amino-9-ethylcarbazole as a substrate (ZYMED Laboratories, San Francisco, CA, USA). Sections were then stained with hematoxylin for counterstaining and mounted using VectaMount AQ Aqueous Mounting Medium (Cat-No. H-5501; Vector Laboratories). Myeloperoxidase-positive (Abcam), F4/80-positive (Santa Cruz, 377009), and LY6C-positive cells (Abcam, ab15627) were counted in stained sections in six randomly chosen fields (×200), and bars are indicated standard errors of the means.
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