I assume that the fresh tissue was fixed in methanol? If so, it is irreversibly coagulant- fixed ( like boiling an egg white...no going back) Place the specimen in dist water until it melts to RT x3 changes of dw 1hr each at RT on a gentle rocker to get rid of methanol ( antifreeze, hence inability to freeze as you have replaced water) Place in OCT at RT on a rocker for 30 mins Orientate and snap-freeze Or, if your mass spec doesn't like OCT....freeze without OCT ? NB: Most important to freeze quickly to avoid ice-crystal formation Proper snap-freezing is used to make the water go from liquid to vitreous ( glass-like) state, avoiding the ice-crystal state ( which will occur with SLOW freezing....thus giving you horrible ice-crystal artefact) Good luck!
Carl Hobbs FIBMS Histology and Imaging Manager Wolfson SPaRC Guys Campus, London Bridge Kings College London London SE1 1UL 020 7848 6810 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet
