Rhodamine conjugated anti rabbit igg

Growing oocytes collected at postnatal day 7 were fixed in 3.7% paraformaldehyde in PBS for 20 min, washed with PBS containing 0.1% BSA, permeabilized with 0.5% Triton X-100 for 15 min. The cells were denatured with 4 N HCl for 10 min, neutralized with 100 mM Tris-HCl (pH 8.5) for 20 min, and then incubated with 1/500 anti-5mC (Eurogentec) and 1/500 anti-5hmC (Active Motif) primary antibodies for 1 h at room temperature. After washing with in PBS with BSA, the cells were incubated with 1/250 fluorescein isothiocyanate-conjugated anti-mouse IgG (Jackson Immuno-Research) and 1/250 rhodamine-conjugated anti-rabbit IgG (Jackson Immuno-Research) for 1 h. The oocytes were then mounted on a glass slide in VECTASHEILD medium with DAPI (Vector Laboratory) and observed under a CSU-10 confocal laser scanning microscope (Yokogawa) with an ImagEM EM-CCD camera (Hamamatsu).

Human brain microvascular endothelial cells (HBMECs) were seeded onto fibronectin pre-coated 12-well μ-slides (ibidi GmbH, Martinsried, Germany) with a cell density of 8 × 104/mL for overnight culturing. HBMECs were then treated with concentrated conditioned medium from control and knockdown cells for 24 h. To neutralize ANGPT2, the anti-ANGPT2 antibody (10 μg/mL; AF623, R&D) was added to the concentrated conditioned medium for 1 h of incubation, and then this medium was used to treat HBMECs. After culturing, cells were fixed with 4% paraformaldehyde in PBS for 20 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS (PBS-T) at 4 °C for 10 min. After blocking with 4% BSA in PBS at 37 °C for 30 min, cells were stained with anti-human VE-cadherin antibody (#2158, Cell Signaling) and rhodamine conjugated anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). DAPI was used for nucleus staining. Slides were mounted and viewed under a fluorescence microscope (Zeiss Axio Observer Z1) and analyzed using the AxioVision software (Zeiss). The length of VE-cadherin reflected by fluorescence was calculated by the ImageJ program and normalized to the cell number.