Arterial tissue sections (S3 Fig .) were incubated overnight at 4°C with rat anti-mouse CD18 (1:50, clone C71/16, Cedarlane), Cy3-conjugated mouse-anti human α-actin (1:15000, clone 1A4, Sigma-Aldrich), goat anti-mouse/human ApoB (1:100, R&D systems), and rabbit anti-Ki67 (1:100, clone SP6, GeneTex). The sections were washed and incubated with F(ab)2 AF594-conjugated monovalent donkey anti-goat (1:200, Jackson ImmunoResearch,) and biotinylated donkey anti-rabbit (1:200, Jackson ImmunoResearch) at room temperature for 45 min, followed by Atto425-conjugated streptavidin (1:200, ATTO-TEC) and AF488-conjugated goat anti-mouse CD31 (1:150, R&D systems) at room temperature for 45 min. Nuclei were stained with DAPI (2 μg/ml for 4 min) and the slides were mounted with ProlongGold mounting media (Life Technologies).
Clone sp6
Manufactured by GeneTex
Sourced in United States
The Clone SP6 is a laboratory equipment designed for the in vitro transcription of RNA. It facilitates the synthesis of RNA from DNA templates using the SP6 RNA polymerase.
Automatically generated - may contain errors
Lab products found in correlation
Prolong gold mounting media, by Thermo Fisher Scientific (1 mentions)
Atto425 conjugated streptavidin, by ATTO-TEC (1 mentions)
Clone 1a4, by Merck Group (1 mentions)
Biotinylated donkey anti rabbit, by Jackson ImmunoResearch (1 mentions)
Rabbit anti ki67, by GeneTex (1 mentions)
Standard 25 microscope, by Zeiss (1 mentions)
2 protocols using clone sp6
1
Multicolor Immunofluorescence Staining of Arterial Tissue
2
Immunohistochemical Analysis of Mouse Intestinal Neoplasia
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Tissue specimens were fixed in 10% formalin and histological examination was performed on paraffin-embedded sections followed by hematoxylin and eosin (H&E) as well as immunohistochemical staining. Ki67 and HER-2 overexpression analyses were conducted on 4-µm-thick tissue sections. A primary monoclonal rabbit antibodies against the Ki67 (Clone SP6; GeneTex, CA, USA) and the ErbB2/Her2 (SP3) proteins (Clone SP3; Novus Biologicals, CO, USA) were used. Slides were examined by standard light microscopy using a Standard 25 microscope (Carl Zeiss, Germany) and images were taken with a Canon G9 color camera coupled to the microscope.
The histologic classification was staged with the recommended nomenclature for intestinal neoplasia in mouse models 18 (link). The nomenclature parallels that used for humans. For immunohistochemical HER-2 analysis, membrane was evaluated on a score of 0 to 3+ using the score system suggested by Hoffman et.al.19 (link). For Ki67, nuclear cell staining is taken as a positive reaction, and the results were expressed as percentage of positive cells of 100 counted cells. The results were grouped in 4 groups according to the percentage of stained cells: (1) <=10%; (2) 11% to 30%; (3) 31% to 50%; and (4) >50% positive cells.
Sections were examined separately by two independent pathologists, without any prior knowledge of each experimental group.
The histologic classification was staged with the recommended nomenclature for intestinal neoplasia in mouse models 18 (link). The nomenclature parallels that used for humans. For immunohistochemical HER-2 analysis, membrane was evaluated on a score of 0 to 3+ using the score system suggested by Hoffman et.al.19 (link). For Ki67, nuclear cell staining is taken as a positive reaction, and the results were expressed as percentage of positive cells of 100 counted cells. The results were grouped in 4 groups according to the percentage of stained cells: (1) <=10%; (2) 11% to 30%; (3) 31% to 50%; and (4) >50% positive cells.
Sections were examined separately by two independent pathologists, without any prior knowledge of each experimental group.
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