i am having trouble breaking the nuclear membrane when doing these metaphase spreads. i use 0.56% hypotonic oslution for 15min in the incubator followed by 3x wash with Carnoys fixative (3:1 methanol:acetic acid) for 30min eahc wash and spin at around 2500rpm. Does aging the cells, ex leaving them longer in carnoys in the -20 freezer make cells more brittle? should i put them in the incubator wiht carnoys and then chill them with carnoys again?
thanks