Immunofluorescence. MEF and HeLa cells were plated on glass coverslips and fixed with 4% (v/v) paraformaldehyde/PBS at room temperature for 10 min (or as detailed) prior to immunofluorescence staining. Primary and secondary Angiotensin (1-7) were diluted in 0.5% BSA dissolved in PBS and supplemented with 0.2% saponin. Incubations were done at room temperature for 1 h with primary antibodies, followed by 30 min. with secondary antibodies. All confocal images were acquired with a Zeiss LSM 5 Pascal microscope (Carl Zeiss, Thornwood, NY), using a 63× 1.4 numerical aperture objective.
Filipin staining of free cholesterol. Filipin staining was done essentially as described [15], with minor modifications. Briefly, fixed MEF cells were stained with 2 μg/ml Filipin (final concentration) for 1 h at room temperature. Unbound Filipin was washed extensively with PBS. Autofluorescence of Filipin was detected by excitation with a 405 nm laser, and collecting emission with a 420–480 nm band pass filter [31] and [32].
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