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).
Goat anti fitc alexa fluor 488
Manufactured by Thermo Fisher Scientific
Goat anti-FITC Alexa Fluor 488 is a secondary antibody that binds to fluorescein isothiocyanate (FITC)-labeled primary antibodies or proteins. It is conjugated with the Alexa Fluor 488 fluorescent dye, which emits green fluorescence when excited by a suitable light source. This product can be used in various immunoassay and imaging techniques.
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Lab products found in correlation
2 protocols using goat anti fitc alexa fluor 488
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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