Alexa fluor 488 conjugated rabbit anti mouse igg

After fixing with 5% formaldehyde, the eyes were embedded in paraffin and sectioned at 5-μm thickness. Following deparaffinization and rehydration, sections were hematoxylin and eosin (H&E) stained. The images were collected by Nikon Eclipse light microscope (Nikon Corporation). For immunohistochemistry, the sections were treated with Target Retrieval Solution (DakoCytomation) at 120 °C for 10 min. After blocking, they were incubated with anti-CD31 antibody (1:50; Santa Cruz Biotechnology) overnight at 4 °C and then were incubated with Alexa Fluor 488 conjugated rabbit anti-mouse IgG (1:500; Life Technologies) and 4′6-diamidino-2-phenylindole (DAPI). Slides were mounted with Ultramount Aqueous Permanent Mounting Medium (DakoCytomation) and observed with a confocal fluorescence laser microscope (LSM 700).

Rat aortic smooth muscle cells (RASMCs) were obtained from the thoracic aortas of 2 month-old Winstar rats, as described [44 (link)]. Experiments were performed in adherence with the Institutional Guidelines on the Use of Laboratory Animals. Cells were grown at 37 °C with 5% CO₂ in DMEM containing 10% FBS, 100 units/ml penicillin, and 100 μg/ml streptomycin. To confirm the identity of isolated cells, immunofluorescent staining against α-SM-actin was performed. Cells were fixed in methanol and blocked in PBS 1X + 3% BSA and incubated with α smooth muscle actin primary antibody (Novus Biologicals, NB600-531, Littleton, CO, USA, dilution 1:200) for 1 h at room temperature followed by immunoreaction with FITC-conjugated goat anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA, USA, dilution 1:1000). Cells were also stained with VE-Cadherin antibody and then treated with Alexa Fluor 488-conjugated rabbit anti-mouse IgG (Life Technologies, Carlsbad, CA, USA, dilution 1:1000) to exclude endothelial cells contamination. Nuclei were stained with DAPI (Life Technologies, Carlsbad, CA, USA). Stained cells were analysed by immunofluorescent microscope (40× objective).

Cells were fixed with PBS containing 4% paraformaldehyde for 10 min at room temperature and washed with PBS. Continuously, cells were treated with PBS containing 5% normal goat or rabbit serum (Nichirei Biosciences Inc., Tokyo, Japan) and 0.1% Triton X-100 at room temperature. Then cells were fixed and stained with antibodies to Oct4 (1/200 Millipore, Billerica, MA), Tra-1-60 (1/200, Stemgent^®, Cambridge, MA), Tra-1-81 (1/200, Stemgent^®), SSEA-1 (1/100, Stemgent^®), SSEA-4 (1/100, Stemgent^®), MAP2 (1/200, Millipore), Nestin (1/200, Millipore), α-smooth muscle actin (α-SMA: pre-diluted, DAKO Cytomation, Glostrup, Denmark), α-fetoprotein (1/100, R&D Systems Minneapolis, MN), and to the SeV nucleocapsid protein (mouse monoclonal antibody, clone #2E4, 1.6 mg/mL). These primary antibodies were visualized with Alexa Fluor^® 488-conjugated goat anti-rabbit IgG, or Alexa Fluor^® 488-conjugated rabbit anti-mouse IgG, or Alexa Fluor^® 488-conjugated goat anti-mouse IgG (1/200, Invitrogen, Carlsbad, CA). Cell nuclei were stained with 4', 6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Fluorescence images were taken using a Zeiss inverted LSM 700 confocal microscope (Carl Zeiss, GmbH, Germany). Alkaline phosphate (ALP) staining was performed using a Fast Red substrate kit (Nichirei) according to manufacturer’s instruction.