overnight with anti fx polyclonal antibodies raised in our laboratory diluted 1 2000 and then washed 3 u00d7 10 min with tween 20 buffer and incubated for 45 min with hrp conjugated secondary goat antibody against anti rabbit igg at room temp finally the nitrocellulose strips were washed 3 u00d7 10 min with tbs tween the bands were visualized by chemoluminescence ecl reaction and autoradiography by exposure to kodak x ar5 film eastman kodak co rochester n y u s a for 1 8 min the total amount of protein in the lanes was verified by immunoblotting the membrane with anti actin antibodies diluted 1 2000 cultured iuc cells grown as monolayers were harvested by a 10 min incubation with h h medium hanks balanced salt solution hbss sigma israel chemicals ltd rehovot israel containing bsa 2 mg ml fraction v sigma and 10 mm hepes ph 7 4 supplemented with 5 mm edta at 37 washed cells were resuspended in the same medium at a concentration of 1 u00d7 10 7 cells ml and kept at room temperature until use cells were diluted 20 fold into binding medium h h supplemented with 2 mm ca 2 and immediately perfused through the flow chamber an e selectin coated substrate was prepared as described fuhlbrigge et al j cell biol 135 837 848 1996 briefly protein a 20 u03bcg ml in coating medium sigma was spotted onto a polystyrene plate the substrate was blocked with 2 human serum albumin hsa fraction v calbiochem la jolla calif u s a in pbs and overlaid with culture supernatant from cos cells.