X gal

X-gal staining for β-galactosidase activity was performed on HCEC treated with rapamycin 2 nM and control (DMSO) at week two and week five. Each sample was rinsed twice with PBS, fixed in 3.7% PFA for 15 minutes. Cells were washed with PBS, and then stained for 36 hours at 37 °C in X-gal solution with PH of 6.0 containing 1 mg/ml X-gal (Thermo Scientific), 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl₂. X-gal positive cells (cells with greenish color) were examined in 10 fields from each sample under light microscope (Carl Zeiss, Thornwood, NY), and photographed with an AxioCam (Carl Zeiss) camera.

Chinese hamster ovary (CHO-K1) cells grown in a 6-well plates were transfected with 2.5 µg of human or ZF encoded 3-OST isoforms (3-OST-2,or 4), or pCDNA3.1 using LipofectAMINE (Gibco/BRL). At 16 h posttransfection, the cells were replated into 96-well dishes for infection with recombinant virus. After 6-h post infection, β-galactosidase assay were performed using either a soluble substrate o-nitrophenyl-β-D-galactopyranoside (ONPG; ImmunoPure, Pierce) or X-gal (Sigma). For the soluble substrate, the enzymatic activity was measured at 410 nm using a micro-plate reader. For X-gal assay, the cells were fixed (2% formaldehyde and 0.2% glutaraldehyde) and permeabilized (2 mM MgCl2, 0.01% deoxycholate, and 0.02% nonidet NP-40 (Sigma). Finally, 1 mL of β-galactosidase reagent (1.0 mg/ml X-gal in ferricyanide buffer) was added to each well and incubated at 37°C for 90 min before the cells were examined using bright field microscopy under the 20 × objective (Zeiss, Axiovert 100M). Heparinase-II and -III were obtained from Sigma–Aldrich Chemical.