Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans

Cell monolayers were fixed at the indicated time posttransfection by washing three times with PBS and incubating for 15 min at 37'C in a 3.7 % solution of formaldehyde in PBS. Coverslips were removed from the wells, washed with PBS, and permeabilized by incubation for 1 min in a solution of 100% methanol at -20°C . After washing with PBS the cells were incubated in a solution containing 1% FCS in PBS for 30 min at room temperature followed by a 30-min incubation at 30°C with a 1:2,000 dilution of anti-Man II antibody (27) in PBS. The coverslips were washed again in PBS and incubated for 30 min at 30°C with a 1 :1,000 dilution of a FITC-conjugated goat anti-rabbit antibody (Cappel Laboratories) in PBS containing 1% FCS. Cells were observed and photographed using an Axioplan microscope (Carl Zeiss, Oberkochen, Germany) .