Human aortic samples were fixed in 10% formalin, embedded in paraffin, and sectioned at 5 μm intervals. Immunohistochemical staining was performed using established methods [17 (link)]. To remove the paraffin, sections were treated with xylene and rehydrated. Then, they were incubated with 3% H2O2 for 10 min at room temperature and washed 3 times with phosphate-buffered saline (PBS). After blocking with serum for 30 min, the sections were incubated with primary antibodies against YAP (1 : 1000 dilution, Cell Signaling), α- smooth muscle actin (α-SMA, 1 : 500 dilution, Sigma), Bcl-2 (1 : 1000 dilution, Cell Signaling), and cleaved caspase-3 (1 : 300 dilution, Cell Signaling), followed by incubation with the ChemMate EnVision System (Dako). ImageProPlus 3.0 (ECIPSE80i/90i) was used to capture the images and analyze the results. For cryostat sections, human and mouse aortic samples were fixed in 4% paraformaldehyde, embedded in optimum cutting temperature (OCT) compound, frozen in liquid nitrogen, and sectioned at 5 μm intervals. DeadEnd Fluorometric TUNEL (Promega) was used to detect the apoptotic cells. Apoptotic VSMCs were detected by TUNEL and α-SMA double staining before confocal fluorescence microscopy analysis (Leica Microsystems).
Chemmate envision system
Manufactured by Agilent Technologies
Sourced in Denmark
The ChemMate EnVision system is a versatile laboratory equipment designed for automated sample preparation and analysis. It features an integrated liquid handling system and a modular design to accommodate various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
Deadend fluorometric tunel, by Promega (1 mentions)
α sma, by Leica Microsystems (1 mentions)
α smooth muscle actin α sma, by Merck Group (1 mentions)
Bcl 2, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Hyaluronidase type 1 s, by Merck Group (1 mentions)
Envision, by Agilent Technologies (1 mentions)
Mac 2, by Santa Cruz Biotechnology (1 mentions)
Elastic fiber staining kit, by Maixin Group (1 mentions)
Deadend fluorometric tunel kit, by Promega (1 mentions)
Envision plus, by Agilent Technologies (1 mentions)
Anti cav1, by Cell Signaling Technology (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Trypsin type 2, by Merck Group (1 mentions)
Peroxidase dab kit, by Agilent Technologies (1 mentions)
Phospho p38, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
8 protocols using chemmate envision system
1
Immunohistochemical Analysis of Aortic Tissue
2
Immunohistochemical Analysis of Paraffin Sections
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Immunohistochemical analysis of paraffin sections was performed using the ChemMate EnVision system (Dako) according to the manufacturer's instructions. Deparaffinized and rehydrated sections were immersed in 0.3% hydrogen peroxide in methanol for 30 min at room temperature to block endogenous peroxidase activities. For SOX9, K17 and Ki-67 immunostaining, antigen retrieval was performed by first autoclaving sections in citric acid buffer (pH 6.0) at 121°C for 10 min. To analyze the stromal expression of perlecan, the sections were incubated with hyaluronidase (Type IS, 330 UI/ml; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) for 30 min prior to immunostaining. After antigen retrieval, the sections were incubated with 5% milk protein in phosphate-buffered saline (PBS) for 30 min and then with the primary antibodies overnight at 4°C. After washing with PBS, the sections were reacted with EnVision (Dako) for 60 min at room temperature. Peroxidase reaction products were developed with 3,3′-diaminobenzidine, and the sections were counterstained with hematoxylin. In the control experiments, the primary antibodies were replaced with the appropriate negative control immunoglobulin (cat. no. X0931 for mouse IgG1 and cat. no. X0903 for rabbit Ig fraction; Dako).
3
Immunohistochemistry and Apoptosis Analysis of Mouse Aorta
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Histology and immunohistochemistry staining were performed as described recently [4 (link),5 (link)]. Briefly, mouse aortas were fixed, embedded, and sectioned at 5-μm intervals, and incubated with 3% H2O2 for 10 min. Sections were then blocked with serum and incubated with primary antibodies at 4°C overnight. Primary antibodies against Mac-2 (Santa Cruz Biotechnology, CA; 1:200 dilution), Gr-1 (Abcam, Cambridge, MA; 1:200 dilution), and IgG (Santa Cruz) were used as a negative control. Sections were then incubated with the ChemMate™ EnVision™ System (Dako, Glostrup, Denmark). Images were captured and further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i); additional images are provided in Supplementary Figure S1.
Cell apoptosis was detected using a commercial DeadEnd Fluorometric TUNEL Kit (Promega, Madison, WI). Costaining of Tunel with α-SMA or CD31 was performed to detect apoptotic SMC or apoptotic EC prior to confocal fluorescence microscopy analysis (Leica Microsystems, Buffalo Grove, IL).
HE staining was performed as described elsewhere. Elastin staining involved the use of an Elastic Fiber Staining Kit (Maixin Bio, Fuzhou, China) as described recently [5 (link)]. All images were further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i).
Cell apoptosis was detected using a commercial DeadEnd Fluorometric TUNEL Kit (Promega, Madison, WI). Costaining of Tunel with α-SMA or CD31 was performed to detect apoptotic SMC or apoptotic EC prior to confocal fluorescence microscopy analysis (Leica Microsystems, Buffalo Grove, IL).
HE staining was performed as described elsewhere. Elastin staining involved the use of an Elastic Fiber Staining Kit (Maixin Bio, Fuzhou, China) as described recently [5 (link)]. All images were further analyzed using ImageProPlus 3.0 (ECIPSE80i/90i).
4
Immunohistochemical Analysis of CAV1 Expression
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Immunohistochemistry staining of the paraffin sections was performed using the ChemMate EnVision system (Dako, Glostrup, Denmark). The sections were deparaffinized in xylene and rehydrated in ethanol. Antigens were retrieved by boiling the sections in 0.5 M EDTA (pH 8) using a pressure cooker for 10 min and under high pressure for 3 min. After cooling to RT, endogenous peroxidase was blocked by incubating the sections with 0.3% hydrogen peroxide for 10 min. The sections were incubated with the primary antibody anti-CAV1 (1:400; D46G3 rabbit, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight. After washing with TBS, the sections were incubated with EnVision Plus (Dako, Glostrup, Denmark) at RT for 30 min. The signals were developed using 3, 3′-diaminobenzidine (Dako, Glostrup, Denmark), and the sections were counterstained using hematoxylin.
5
Immunohistochemistry protocol for various markers
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Immunohistochemistry was performed using the ChemMate Envision system (Dako, Glostrup, Denmark), as described elsewhere1 (link)–3 (link). For K10, K13, K17, and K19, sections were pretreated as previously described2 (link),21 (link). For cleaved caspase 3 (caspase 3) and CD68, sections were autoclaved at 121 °C for 10 min in Tris–EDTA buffer (pH 9.0) and citric acid buffer (pH 6.0), respectively. For CD31, sections were pre-treated with 0.2% trypsin (type II, Sigma Chemical Co, St. Louis, MO, USA) in 0.01 M Tris–HCl (pH 7.6) containing 0.1% CaCl2 for 30 min at 37 °C. For the control experiments, the primary antibodies were replaced with pre-immune IgGs. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed as described elsewhere4 (link). K17+ speckles were compared with Civatte bodies seen in serial HE-stained sections.
6
Immunohistochemical Staining of Esophageal Tissues
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Was performed immunohistochemical staining using the rabbit or mouse DAKO ChemMate EnVision system and a Peroxidase/DAB kit (DAKO, Carpinteria, California). The sample containing paraffin was sliced into serial sections with a width of 5.0 μm. Each section was deparaffinized for 1 hour at 60°C in xylene and rehydrated in serial-graded ethanol before being stored overnight in citrate buffer (0.01 M; pH 6.0) at 75°C for antigen retrieval. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol. The sections were incubated at 4°C overnight with a primary antibody and then recovered at 37°C for 20 minutes. The slides were incubated with the secondary antibody for 30 minutes at room temperature and then were developed by DAB. Nuclear staining was carried out with hematoxylin. Phosphate-buffered saline was added as the primary antibody in the negative control. Normal esophageal tissue, serving as the negative controls, and esophageal cancer tissue (known to express PI3K/AKT/mTOR signaling pathway proteins) serving as positive controls were processed in the same way. However, normal esophageal tissue serves as the positive controls, and esophageal cancer tissue serves as negative controls for PTEN.
7
Immunohistochemistry of Extracellular Matrix Proteins
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Tissue sections were deparaffinized, rehydrated, and preincubated with goat serum for 5 minutes. Subsequently, sections were incubated with collagen type I primary antibodies (monoclonal rabbit, anti-human; Cederlane, Hornby, ON, Canada), versican (monoclonal mouse, anti-human 1 [generously provided by Developmental Studies Hybridoma Bank, Iowa City, IA, USA]), decorin (monoclonal mouse, anti-human [generously provided by Developmental Studies Hybridoma Bank]), biglycan (polyclonal rabbit, anti-human [provided by P Roughley, Shriners Hospital, McGill University]), and alpha-smooth muscle actin (α-SMA) (monoclonal mouse, anti-human; Sigma-Aldrich Co, St Louis, MO, USA).
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse (ChemMate EnVision™ System; DakoCytomation, Troy, MI, USA) was used as a secondary antibody. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin. For negative controls, the primary antibody was omitted from the staining.
Horseradish peroxidase-conjugated anti-rabbit or anti-mouse (ChemMate EnVision™ System; DakoCytomation, Troy, MI, USA) was used as a secondary antibody. Diaminobenzidine was used as the chromogen. The sections were counterstained with hematoxylin. For negative controls, the primary antibody was omitted from the staining.
8
Immunohistochemical Analysis of Signaling Pathways
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Immunohistochemical staining was performed on 5-μm sections of the paraffin-embedded tissue blocks. Paraffin sections were mounted on superfrost glass slides, deparaffinized in xylol, rehydrated in a graded ethanol series, and then subjected to microwave antigen retrieval (3 × 10 min, 800w, in 0.1 M citrate acid buffer solution, pH 6). Endogenous peroxidase activity was blocked using 0.3% hydrogen peroxide. Sections were incubated at 4°C overnight with the following primary antibodies at the specified dilutions: S100 (DAKO Z0311, dilution 1:100), phospho ERK1/2 (Cell Signaling Technology #4370 dilution 1:100), phospho p38 (Cell Signaling Technology #4511, dilution 1:50), and LC3a/b (Cell Signaling Technology #, dilution 1:50). Immunohistochemical staining was performed using the ChemMateEnVision system (DAKO K5007) using the suggested manufacturer protocol. Sections were then counterstained with Mayer hematoxylin, dehydrated, cleared, and mounted. Appropriate positive controls were used according to the manufacturer's guide for each antibody.
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