Frozen 5μm aortic roots were fixing for 10 min in acetone (Sigma-Aldrich) at RT and placed into 1% paraformaldehyde in 100mM sodium phosphate containing 60mM lysine and 7mmM sodium periodate (Sigma-Aldrich) pH 7.4 on ice. Then slides were permeabilized with freshly prepared 0.1% Triton X-100, 0.1% sodium citrate solution and incubated with TUNEL reaction mixture for 60 minutes at 37°C in the dark. The sections were i ncubated with avidin/biotin blocking kit (Vector laboratories) followed by incubation in a 5% normal goat serum and 1% BSA (Sigma) in PBS. Aortic root sections were stained overnight at 4°C with primary antibodies: rat anti-CD11bFITC (M1/70, BD Bioscience) followed by staining with secondary antibodies for 1 h at RT: goat anti-FITC Alexa Fluor 488 (Molecular Probes).
Rat anti cd11bfitc m1 70
Manufactured by BD
Rat anti-CD11b FITC (M1/70) is a fluorescently labeled monoclonal antibody that specifically binds to the CD11b antigen expressed on the surface of myeloid cells, including monocytes, macrophages, and granulocytes. This antibody can be used for the identification and characterization of these cell populations in flow cytometry applications.
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Lab products found in correlation
Goat anti fitc alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Sodium periodate, by Merck Group (1 mentions)
Avidin biotin blocking kit, by Vector Laboratories (1 mentions)
Acetone, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Rat anti cd3 17a2, by BioLegend (1 mentions)
2 protocols using rat anti cd11bfitc m1 70
1
TUNEL Assay for Apoptosis in Aortic Tissue
2
Immunofluorescent Staining of Aortic Root Sections
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Immunofluorescent staining of 5 μm aortic root section was performed as previously described19 (link). Briefly, aortic sections were stained overnight at 4 °C with primary antibodies specific to mouse antigens: hamster anti-CD11c (HL3, BD Bioscience), rat anti-CD11b-FITC (M1/70, BD Bioscience), rat anti-CD3 (17A2, Biolegend) followed by staining with secondary antibodies for 1 hour at room temperature (RT): goat anti-FITC Alexa Fluor 488 (Molecular Probes), goat anti-rat IgG Alexa Fluor 568 (Molecular Probes), and goat anti-hamster IgG DyLight 649 (Jackson Immunoresearch). MHCII was stained by anti-MHCII-PE (M5/114.15.2; eBioscience) Sections were counterstained with DAPI and embedded in Prolong Gold. Images were acquired on a Leica SP8 DM6000 inverted confocal microscope using HCX PLAPO 20x and 40x oil-immersion objectives at 405 nm, 488 nm, 563 nm and 633 nm excitation wavelength. Imaris Software was employed to adjust brightness and one-step smoothing on all images in parallel.
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