Fitc goat anti rat igg

The cross-section samples of the aortic sinus were used for immunofluorescence. The slides were blocked in 10% goat serum diluted with PBS for 30 min and incubated overnight at 4 degrees Celsius with various primary antibodies: LC3 (L7543, Sigma, USA), p62 (PM045, MBL, Japan), CD11b (553312, BD, USA), TFEB (13372-1-ap, proteintech, China), LAMP1 (EPR21026, abcam, UK). After rewarming at room temperature for 10 min, the sections then were incubated with the relevant secondary antibody for another 1 h. The secondary antibodies used were FITC goat anti-rabbit IgG (Invitrogen, USA), TRITC goat anti-rabbit IgG (Invitrogen, USA), FITC goat anti-rat IgG (Invitrogen, USA). The nuclei were stained with 0.5 g/L DAPI for 10 min. Images were obtained using a Confocal microscope (ZEISS LSM 800) and the images were analyzed with Image Pro Plus 6.0.
Cells were seeded onto glass slides. After the indicated treatments, cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X-100. Subcequently, cells were incubated with primary antibodies and secondary antibody as described in histochemistry, cells fluorescence intensity was observed under Confocal microscope (ZEISS LSM 800) and representative cells were selected and photographed. Quantification analysis was performed using ImageJ.

Cells were seeded at a density of 23.500 cells/cm² and allowed to attach overnight. Next, cells were exposed to normoxia (21% O₂) or moderate hypoxia (0.2% O₂) for 24 h. Subsequently, BrdU was added to the culture medium (10 μM) for 1 h after which cells were fixed in 4% paraformaldehyde for 20 min. After permeabilization with 0.1% triton X‐100 for 20 min, the cells were treated with 2 M HCl for 30 min at 37°C and twice with 0.1 M borate for 5 min. After blocking in 10% FCS for 30 min, cells were incubated with primary antibody (anti‐BrdU, ABD serotec, OBT0030S) 1/50 for 90 min and with secondary antibody (FITC‐Goat Anti‐Rat IgG, Invitrogen, 62‐9511) 1/200 for 1 h. Nuclei were stained with Hoechst for 10 min. Images were taken using the Nikon Eclipse Ts2 fluorescent microscope. The amount of (BrdU‐positive) nuclei was quantified using Image J software and cell proliferation was expressed as the percentage of BrdU positive nuclei from the total amount of nuclei.