After exposure of HAoECs to one cycle of IH, cells were incubated (1 hour, room temperature) in blocking buffer (1% non-fat dry milk in physiological buffer solution). After 3 washes, cells were incubated overnight (4 °C) with anti-IL-6 and anti-VAMP (1:100, Santa Cruz Biotechnology) antibodies. Three washes were followed by incubation (2 hours, room temperature) with secondary mouse fluorescence-labelled antibody Fluoprobes FP 546 and secondary rabbit fluorescence-labelled antibody Fluoprobes 488 (1:500, Interchim, San Diego, CA). Images were then taken using confocal microscope and analyzed with Zeiss software.
Fluoprobes fp 546
Manufactured by Interchim
The Fluoprobes FP 546 is a fluorescent probe designed for various analytical applications. It exhibits excitation and emission wavelengths suitable for many fluorescence-based techniques.
Automatically generated - may contain errors
2 protocols using fluoprobes fp 546
1
Immunofluorescence analysis of IL-6 and VAMP in HAoECs
2
Immunostaining for Vascular Inflammation
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For immunostainings, aorta and carotid sections were fixed in 4% paraformaldehyde. Nonspecific binding was blocked with 5% nonfat dried milk in PBS, pH 7.4. Sections were incubated overnight at 4°C with monoclonal anti–inducible NOS (1:100; BD Biosciences, Le Pont de Claix, France), anti–NF‐κB (1:100; Abcam, Cambridge, UK), and anti‐F4/80 (1:100; Biolegend, San Diego, CA) antibodies. After 3 washes, the primary antibodies were detected after 1‐hour incubation with a secondary mouse or rabbit fluorescence‐labeled antibody Fluoprobes FP 546 (1:100; Interchim, San Diego, CA).
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