Cells were seeded on glass coverslips precoated with collagen in 24-well plates. After incubation at 37°C, cells were treated according to the experimental protocol with E2 (200 nM), raloxifene (20μM), and S (10 ng/ml). After 72 hours, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS, and stained overnight at 4°C with ACE2 protein–specific antibody (Abcam, Ab15348). Cells were then incubated with anti-rabbit secondary antibody (Alexa Fluor 536 anti-rabbit, Invitrogen Life Technologies) for 1 hour at 37°C. Nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific) for nuclear staining for 20 min. Cells were mounted with Fluor mount (Sigma-Aldrich, St. Louis, MO, USA), and images were acquired through confocal microscope LSM 800, 60× magnification, software ZEN 2.1 blue edition (Carl Zeiss, Jenza, Germany) and analyzed with ImageJ software.
Alexa fluor 536 anti rabbit
Manufactured by Thermo Fisher Scientific
Alexa Fluor 536 anti-rabbit is a fluorescent secondary antibody conjugate designed for use in immunological applications. It is generated by conjugating the Alexa Fluor 536 dye to an anti-rabbit antibody. The Alexa Fluor 536 dye has an excitation maximum of 536 nm and an emission maximum of 555 nm, making it suitable for detection in the green-yellow region of the visible spectrum.
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2 protocols using alexa fluor 536 anti rabbit
1
Measuring ACE2 Expression in Cells
2
ACE2 Expression in E2 and Raloxifene Treated Cells
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Cells were seeded on glass coverslips pre-coated with collagen in 24-well plates. After incubation at 37°C, cells were treated according to the experimental protocol with E2 (200nM), Raloxifene (20μM), S (10ng/ml). After 72 hours, cells were washed, fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100 in PBS and stained overnight at 4°C with ACE2 protein-specific antibody (Abcam Ab15348). Cells were then incubated with anti-rabbit secondary antibody (Alexa Fluor 536 anti-rabbit, Invitrogen Life Technologies) for 1 hour at 37°C. Nuclei were labeled with Hoechst 33342 (Thermo Fisher Scientific) for nuclear staining for 20 minutes.
Cells were mounted with Fluor-mount (Sigma-Aldrich, St Louis, MO, USA) and images were acquired through confocal microscope LSM 800, magnification 60X, software ZN 2.1 blue Edition (Carl Zeiss, Jenza, Germany) and analyzed with ImageJ software.
Cells were mounted with Fluor-mount (Sigma-Aldrich, St Louis, MO, USA) and images were acquired through confocal microscope LSM 800, magnification 60X, software ZN 2.1 blue Edition (Carl Zeiss, Jenza, Germany) and analyzed with ImageJ software.
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