Histologic sections (3 μm thickness) were prepared from paraffin embedded aortic segments. Representative cross sections were stained with hematoxylin and eosin for the assessment of aortic structure and plaque visualization. Additional unstained slides were used for immunohistochemistry staining for MAC387 (macrophage marker, Abcam ab22506, 1:50 overnight at 4°C), VCAM-1 (vascular cell adhesion molecule-1, Abcam ab98954, 8 μg/ml overnight at 4°C) and p-selectin (CD62p, Bioss bs-0561R, 8 μg/ml overnight at 4°C). Antigen retrieval was performed by heat treatment in Tris EDTA pH 9.0 (for MAC387 staining) or 10 mM Sodium Citrate, 0.05% Tween 20, pH 6.0 (for p-selectin staining) for 20 min using a microwave. Sections were then incubated with a peroxidase labeled polymer (DAKO) as secondary antibody. Staining was performed with chromogen system 3,30-Diaminobenzidine substrate (DAKO) and counterstained in Mayer’s Hematoxylin.
Peroxidase labeled polymer
Manufactured by Agilent Technologies
The Peroxidase labeled polymer is a reagent used in various immunohistochemical and in situ hybridization procedures. It is composed of a polymer backbone with covalently attached peroxidase enzymes. The primary function of this product is to provide a sensitive and efficient means of detecting target proteins or nucleic acid sequences in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Ab22506, by Abcam (1 mentions)
Ab 98954, by Abcam (1 mentions)
Bs 0561r, by Bioss Antibodies (1 mentions)
Rabbit anti mouse cd31, by Abcam (1 mentions)
3 3 diaminobenzidine (dab), by Agilent Technologies (1 mentions)
Axiovert microscope, by Zeiss (1 mentions)
Apoptag fluorescein in situ apoptosis detection kit, by Roche (1 mentions)
Donkey anti mouse amca, by Jackson ImmunoResearch (1 mentions)
Donkey anti rabbit 488, by Jackson ImmunoResearch (1 mentions)
Somatostatin, by Abcam (1 mentions)
Biotinylated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
4 protocols using peroxidase labeled polymer
1
Immunohistochemical Analysis of Aortic Plaque
2
Immunohistochemical Quantification of Angiogenesis
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After fixing in 10% formalin, prostate samples were embedded in paraffin and cut at 4 µm thickness. The sections were then incubated with primary antibody (rabbit anti-mouse CD31; 1:50; Abcam Co.) overnight at 4° C followed by a 2 h staining with a peroxidase-labeled polymer conjugated to goat anti-rabbit immunoglobins (Dako Australia Pty, Ltd). The reaction was developed with 3,3’-diaminobenzidine (DAB; Dako Australia Pty, Ltd) and finally counterstained with hematoxylin [24 (link)]. Quantification of CD31+ staining was performed using light microscopy. Sections were analysed on digitised colour images and evaluated by counting the number of positive staining vessels in five fields within the tumour, chosen at random. Vessels were defined as any brown-staining (DAB immunoperoxidase stain with anti-CD31 antigen) cell cluster clearly separated from any adjacent vessels [7 (link)].
3
Immunohistochemical Analysis of Liver Macrophages
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For immunohistochemical analyses, livers (four per group) were quickly removed and fixed
in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH 7.4) for 12 h at 4°C.
For cryoprotection, liver tissue was transferred into graded sucrose (10–30% in 0.1 M PBS,
pH 7.4). The livers were frozen in 2-methyl butane and kept at −80°C until sectioning on a
cryotome. Liver sections (25 µm thick) were collected serially, mounted
on Superfrost glass slides, dried for 2 h at room temperature, and stored at −20°C until
staining. Slides were incubated with appropriate dilution of mouse monoclonal ED1 antibody
1:100 (Abcam, Cambridge, UK ), raised against rat lysosomal membrane antigens of activated
macrophages for 60 min [29 (link)]. Afterwards, slides
were incubated with the peroxidase-labeled polymer (DakoCytomation) conjugated to goat
anti-mouse immunoglobulins for 30 min. The immunoreaction products were visualized with
3′3-diaminobenzidine (DAB, Dako, Glostrup, Denmark) according to the manufacturer’s
instructions. After dehydration and clearing, sections were mounted with mounting medium
DPX (Sigma-Aldrich, St. Louis, MO, USA) and examined under a Zeiss Axio Vert microscope
(Zeiss, Gottingen, Germany).
in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH 7.4) for 12 h at 4°C.
For cryoprotection, liver tissue was transferred into graded sucrose (10–30% in 0.1 M PBS,
pH 7.4). The livers were frozen in 2-methyl butane and kept at −80°C until sectioning on a
cryotome. Liver sections (25 µm thick) were collected serially, mounted
on Superfrost glass slides, dried for 2 h at room temperature, and stored at −20°C until
staining. Slides were incubated with appropriate dilution of mouse monoclonal ED1 antibody
1:100 (Abcam, Cambridge, UK ), raised against rat lysosomal membrane antigens of activated
macrophages for 60 min [29 (link)]. Afterwards, slides
were incubated with the peroxidase-labeled polymer (DakoCytomation) conjugated to goat
anti-mouse immunoglobulins for 30 min. The immunoreaction products were visualized with
3′3-diaminobenzidine (DAB, Dako, Glostrup, Denmark) according to the manufacturer’s
instructions. After dehydration and clearing, sections were mounted with mounting medium
DPX (Sigma-Aldrich, St. Louis, MO, USA) and examined under a Zeiss Axio Vert microscope
(Zeiss, Gottingen, Germany).
4
Immunohistochemical Analysis of Pancreas
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Pancreas samples were fixed in formaldehyde and embedded in paraffin for subsequent analysis. Five-micrometer sections were stained with hematoxylin and eosin or by immunohistochemistry for islet hormones using a cocktail of antibodies to insulin, glucagon, or somatostatin (21 (link)). In addition, sections were immunostained for insulin, Ki67, or DAPI (nucleus) to assess proliferation, TUNEL for apoptosis, and for duct marker using anti-CK19 antibodies, and GLP-1 to identify incretin immunoreactivity. The hematoxylin and eosin slides were examined in all cases by two pathologists to exclude those with pancreatitis, autolysis, and tumor infiltration.
Primary antibodies included insulin (guinea pig antibody, 1:200; Abcam), glucagon (mouse mono, 1:500; Sigma-Aldrich), somatostatin (rabbit poly, 1:500; Abcam), or Ki67 (mouse mono antibody, 1:50; BD Biosciences), GLP-1 (rabbit antibody, 1:1000; J.F. Habener, MD, Massachusetts General Hospital, Boston, MA), and CK19 (rabbit poly, 1:100; Abcam). For TUNEL we used an Apoptag Fluorescein in situ apoptosis detection kit (Roche) and PDX1 (rabbit poly, 1:200; Cell Signaling).
Secondary antibodies were donkey anti–GP-594, donkey anti–mouse-AMCA, donkey anti–rabbit-488, donkey anti–mouse-AMCA, and biotinylated donkey anti-rabbit (all from Jackson ImmunoResearch, West Grove, PA), and peroxidase labeled polymer (Dako).
Primary antibodies included insulin (guinea pig antibody, 1:200; Abcam), glucagon (mouse mono, 1:500; Sigma-Aldrich), somatostatin (rabbit poly, 1:500; Abcam), or Ki67 (mouse mono antibody, 1:50; BD Biosciences), GLP-1 (rabbit antibody, 1:1000; J.F. Habener, MD, Massachusetts General Hospital, Boston, MA), and CK19 (rabbit poly, 1:100; Abcam). For TUNEL we used an Apoptag Fluorescein in situ apoptosis detection kit (Roche) and PDX1 (rabbit poly, 1:200; Cell Signaling).
Secondary antibodies were donkey anti–GP-594, donkey anti–mouse-AMCA, donkey anti–rabbit-488, donkey anti–mouse-AMCA, and biotinylated donkey anti-rabbit (all from Jackson ImmunoResearch, West Grove, PA), and peroxidase labeled polymer (Dako).
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