rabbit antibody 30 mins at room temperature jackson immuno research cambridge great britain or with the alexa fluor u00ae 488 conjugated goat anti mouse igg secondary antibody invitrogen and 0 4 u03bcg ml of dapi 5 u00d7 10 5 msc were plated out into 6 well vessels with very low adherence costar cellsystems and cultured for 4 days in mem which contained 1 mm monothioglycerol 15 es cult fbs and 4 5 g l of dmem high glucose cellsystems the resulting cell clusters were then cultured for 6 days in 6 well adherent plates falcon in its supplemented serum free medium cellsystems after transfer into poly l ornithine coated 24 well plates the cells were cultured for 6 days in a pancreatic proliferation medium cellsystems which contained n2 a and b27 supplement substances and 25 ng ml of rh bfgf and then for a further 6 days in an rh bfgf free pancreatic differentiation medium cellsystems containing 10 mm nicotinamide after washing the cells were fixed with 4 pfa permeabilized with 70 ethanol and incubated with a blocking buffer which contained 0 25 triton x 100 and 2 fbs next they were labeled overnight with a polyclonal rabbit anti human glucagon antibody 1 75 dilution dako cytomations glostrup denmark or with a polyclonal rabbit anti human insulin antibody 1 200 dilution anta cruz biotechnology and stained with a secondary goat anti rabbit igg cy 3 millipore schwalbach germany and 0 4 u03bcg ml of dapi the monoclonal antibody w8b2 igg1 specificity for htnap was obtained by immunization of.