For immunohistochemistry (IHC) on murine tissues, lung sections were deparaffinized and rehydrated, and then subjected to heat-mediated antigen retrieval with 10 mM sodium citrate buffer (pH = 6.0) at 95°C for 15 min. After blocking of endogenous peroxidase activity with 3% hydrogen peroxide (15 min, RT), sections were blocked with 10% normal goat serum (1 h, RT) followed by blocking of endogenous biotin using an Avidin/Biotin blocking kit (Vector Laboratories, Burlingame, CA, United States). Afterwards, primary antibodies for F4/80 (rat anti-mouse F4/80, clone Cl:A3-1, 1:100, AbD Serotec; Kidlington, United Kingdom), and FR-β (rabbit anti-mouse FR-β, 1:400, Genetex, Irvine, CA, United States) were applied on the specimens and incubated overnight at 4°C. Isotype- and concentration-matched IgGs served as negative controls. Next, biotin-labeled goat anti-rat or anti-rabbit secondary antibodies (all from Vector Laboratories) were applied (30 min, RT). This was followed by incubation with the Vectastain ABC Elite HRP kit for 30 min at RT (Vector Laboratories). Finally, stainings were visualized using 3,3′-diaminobenzidine (DAB) in case of F4/80, or 3-amino-9-ethylcarbazole (AEC) (all from Vector Laboratories) in case of FR-β, and sections were counterstained with Mayer's hematoxylin (J.T. Baker, Deventer, Netherlands).
3 amino 9 ethylcarbazole aec
Manufactured by Vector Laboratories
Sourced in United States
3-amino-9-ethylcarbazole (AEC) is a laboratory reagent commonly used as a chromogen in immunohistochemical and enzyme-linked immunosorbent assay (ELISA) applications. It produces a brown-colored reaction product when catalyzed by peroxidase enzymes, allowing for the visualization and detection of target molecules in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Axiocam digital camera, by Zeiss (6 mentions)
Axioplan 2 microscope, by Zeiss (3 mentions)
Rabbit anti st2 polyclonal antibody, by Thermo Fisher Scientific (2 mentions)
Vectastain elite abc kit, by Vector Laboratories (2 mentions)
Vectastatin elite abc kit, by Vector Laboratories (2 mentions)
Ihc kit, by Enzo Life Sciences (2 mentions)
Vectastain elite abc hrp kit, by Vector Laboratories (2 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
3 3 diaminobenzidine (dab), by Vector Laboratories (1 mentions)
Rabbit anti mouse fr β, by GeneTex (1 mentions)
Bz x710 microscope, by Keyence (1 mentions)
Dako envision kit, by Agilent Technologies (1 mentions)
Citrate buffer, by Thermo Fisher Scientific (1 mentions)
Mouse anti cd68, by Novus Biologicals (1 mentions)
Vectastain abc system, by Vector Laboratories (1 mentions)
Hematoxylin qs, by Vector Laboratories (1 mentions)
Mmp12, by Proteintech (1 mentions)
Citrate buffer ph 6, by Thermo Fisher Scientific (1 mentions)
Rabbit igg isotype control, by Santa Cruz Biotechnology (1 mentions)
Rat igg2b, by BD (1 mentions)
16 protocols using 3 amino 9 ethylcarbazole aec
1
Immunohistochemical Analysis of Murine Lung Tissues
2
Immunohistochemical Analysis of VEGF, PECAM, and ICAM-1
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Five-μm sections were dewaxed and rehydrated. After quenching endogenous peroxidase, achieving antigen retrieval and blocking nonspecific binding sites, sections were incubated with the anti-human VEGF-A mouse monoclonal antibody (Beckton Dickinson), at a concentration of 5 μg/ml, anti-mouse PECAM/CD31 rabbit polyclonal antibody (Abcam), at a concentration of 40 μg/ml and anti-mouse CD-54/ICAM-1 hamster monoclonal antibody (BD Pharmingen) at a concentration of 1.25 μg/ml. Secondary biotinylated monoclonal Abs and staining kits were obtained from Vector Laboratories. Immunoreactivity was visualized with peroxidase reaction using 3-amino-9-ethylcarbazole (AEC, Vector Laboratories, Burlingame, CA) in H2O2 and specimen counterstained with hematoxylin. As a negative control, primary Abs were omitted or replaced with an irrelevant isotype-matched mAb. Stained sections were analyzed with the AxioCam digital camera attached to the Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany). VEGF-A staining intensity was evaluated by a semiquantitative, four-stage scoring system, ranging from negative (0) to strong immunoreactivity (4+). Positive cells for ICAM-1 and CD31 were directly counted. Every quantification was performed on three fields per sample by two independent observers, blinded to the status of the specimens.
3
Quantifying Atherosclerotic Plaque Lipids and Macrophages
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Hearts collected after ice-cold PBS perfusion were fixed in 4% paraformaldehyde phosphate buffer solution at 4 °C overnight and embedded in O.C.T. Compound (Sakura FineTech, Tokyo, Japan). Serial frozen sections (7-μm thickness) of the aortic root were prepared as previously described [17 (link),18 (link)]. Atherosclerotic plaques were investigated in five sets of sections, with each set separated by 70 μm. The sections were stained oil Red O to identify the lipid-rich core. Immunohistochemical staining utilized a rat anti-monocyte/macrophage (MOMA2) antibody (1:300, BMA Biomedicals, Augst, Switzerland) and a Vectastain Elite ABC HRP kit (Vector Laboratories, Burlingame, CA, USA). The bound antibody was visualized by the indirect immunoperoxidase method using 3-amino-9-ethylcarbazole (AEC; Vector Laboratories). Aortic root images were captured using a Keyence BZ-X710 microscope (Keyence Corporation, Osaka, Japan). Oil Red O- and MOMA2-positive areas were analyzed using ImageJ software. For each mouse, the mean values from five independent sections were employed for the analysis.
4
TKTL1 Immunohistochemistry in Metastatic Melanoma
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Immunohistochemistry was performed on tissue microarrays constructed from formalin-fixed paraffin-embedded (FFPE) metastatic melanomas and melanoma cell lines after citrate buffer (pH 6.0) antigen retrieval for 30 min using the Dako Envision+ kit (Dako). Incubation with 1× protein blocking buffer for one hour at room temperature was followed by overnight incubation at 4 °C with mouse monoclonal anti-TKTL1 antibody (clone JFC12T10; 1:200 dilution) previously described by Langbein et al. [17 (link)]. 60 min incubation with secondary anti-mouse antibody HRP (Dako) was performed. 3-amino-9-ethylcarbazole (AEC) was used as chromagen (Vector Laboratories). Slides were counterstained with hematoxylin, images were captured and immunohistochemical reactivity was evaluated by two independent investigators. Patient tumor cores were scored TKTL positive based on cytosolic staining in minimum of 10 % tumor cells. Human testis tissue was used as the positive control for TKTL1 and a negative control, for which the primary antibody was substituted with the same concentration of mouse IgG.
5
IHC Protocol for IL-33 and ST2 Detection
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IHC for IL-33 and ST2 were performed with a Vectastain Elite ABC Kit (Vector Lab., Burlingame, CA, USA) according to the manufacturer’s instructions and our published methods [35 (link), 55 (link), 56 (link)]. The following primary antibodies were used: goat anti-IL-33 polyclonal antibody (working dilution 1:100; R&D systems, Minneapolis, MN, USA) and rabbit anti-ST2 polyclonal antibody (working dilution 1:100; Thermo Scientific., Rockford, USA). Antibodies were incubated at 4 °C overnight. 3-Amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA, USA) was used as chromogen, and slides were slightly counterstained with Mayer’s hematoxylin. Previous known colorectal adenoma/carcinoma sections shown to have IL-33 and ST2 IRs were used as positive controls to confirm the IL-33 and ST2 IRs in each series of IHCs. To exclude background staining by nonspecific antibody binding, negative controls were included using isotype-matched antibodies in each IHC test.
6
Immunohistochemical Analysis of ST2 and FoxP3 in Colorectal Cancer
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To examine the expression pattern of ST2-positive cells and FoxP3-positive Tregs in the adenoma/CRC microenvironment, IHC staining was performed on 4-μm paraffin-embedded sections from controls, adenomas and CRC using the Vectastatin Elite ABC Kit (Vector Laboratories Inc, Burlingame, CA, USA) according to the manufacturer’s instructions and our previously published methods4 (link),5 (link). For epitope retrieval, deparaffinized and rehydrated slides were placed in a plastic jar with a boiling solution of 1 mM ethylenediaminetetraacetic acid (EDTA) (pH 8.0) for 20 minutes. The following primary antibodies were used: rabbit anti-ST2 polyclonal antibody (working dilution 1:100; Thermo Fisher Scientific, USA) and mouse anti-FoxP3 monoclonal antibody (working dilution 1:100, Abcam, UK). Tissue sections were incubated with the primary antibodies at 4 °C overnight. Then, the chromogen 3-amino-9-ethylcarbazole (AEC; Vector Laboratories, Burlingame, CA, USA) was used to detect the target antigens, and the tissues were counterstained with Mayer’s hematoxylin. Previous known tumor sections positive for ST2 and FoxP3 were used as positive controls to confirm the immunoreactivity of ST2 and FoxP3 in each series of IHCs. To exclude background staining by nonspecific antibody binding, negative controls were included using isotype-matched antibodies in each IHC test.
7
Immunohistochemical Analysis of Aortic Markers
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Paraffin-embedded aorta sections mounted slides were subjected to heat-induced antigen epitope retrieval with citrate buffer (Thermo Scientific, MA). The slides were incubated overnight at 4°C with primary antibodies for anti—MMP-9 (Rabbit anti-Mouse) (Invitrogen, Carlsbad, CA), Rabbit anti-Mouse anti—MMP-2 (R & D Systems, Minneapolis, MN), Mouse anti- CD-68 (Novus, Cambridge, United Kingdom), anti-Mouse Mac-2 (Cedarlane, Burlington, Canada), and anti-mouse TGFβ1 (R & D Systems, Minneapolis, MN). The sections were incubated with relevant secondary antibodies (Goat anti- Mouse) (Invitrogen, Carlsbad, CA). IHC staining was completed with IHC kit (Enzo Life Sciences, NY). Slides were visualized by 3-Amino-9-ethylcarbazole (AEC) (Vector Laboratories, Burlingame, CA) chromogens followed by an appropriate counterstain.
8
Immunohistochemical Analysis of Melanoma
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Paraformaldehyde-fixed, paraffin-embedded, 4-μ-thick melanoma sections were deparaffinized, rehydrated, and processed for immunohistochemical analysis. The following primary antibodies were used for staining: anti-human CD3 rabbit polyclonal antibody (A0452, Dako, Glostrup, Denmark), at a concentration of 6 μg/mL; anti-human IL-17A goat polyclonal antibody (AF-317-NA, R&D Systems, Minneapolis, MN, USA), at 1:20 dilution; anti-human placenta growth factor (PlGF) rabbit polyclonal antibody (clone 1880, Santa Cruz Biotechnology, Santa Cruz, CA, USA), at 1:50 dilution. Immunoreactivity was visualized with a peroxidase reaction using 3-amino-9-ethylcarbazole (AEC, Vector Laboratories, Burlingame, CA, USA), and the specimens were counterstained with hematoxylin. Negative controls were obtained by omitting the primary antibody. Stained sections were analyzed with an AxioCam digital camera attached to an Axioplan 2 microscope (Carl Zeiss AG, Oberkochen, Germany). Positive cells were counted in 10 fields per specimen at magnification 200×.
9
Immunohistochemical Detection of IL-10
10
Immunohistochemical Detection of Parasite Antigen
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Freshly collected adult parasites were fixed by methacarn fixation and embedded in paraffin. The embedded tissue was cut in 8 μm-sections on a microtome and mounted on gelatin-coated microscopic slides. The parasite sections were deparaffinized twice in xylene and rehydrated in a series of decreasing alcohol concentrations. Antigen retrieval was performed by heating the sections in 10 mM sodium citrate, pH 6.0, 0.05% Tween 20. Non-specific binding sites were blocked by incubation in 1% glycine and 4% BSA in PBS, pH 7.2 each for 30 min at room temperature. After blocking, the parasite sections were incubated with either mouse anti-rOvCALR antiserum or pre-immune serum (1:2,000) in 1% BSA in PBS, pH 7.2 at 4°C overnight. Endogenous peroxidase activity was blocked by incubating sections in 3% H2O2 (Merck, Hohenbrunn, Germany) in the dark at room temperature twice for 10 min. The sections were then incubated in biotinylated polyclonal rabbit anti-mouse immunoglobulin (1:200) (Dako, Copenhagen, Denmark) at 37°C for 1 hr. Following washes, the avidin-biotin complex (ABC) peroxidase staining kit (Thermo Scientific, Rockford, Illinois, USA) and the chromogenic substrate 3-amino-9-ethyl carbazole (AEC) (Vector, Burlingame, California, USA) were used for standard colorimetric detection. The reaction was stopped by washing in PBST (10 mM PBS, pH 7.2, 0.1% Tween 20).
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