Hi, One mm thick is tiny, 2 mm small 1 cm is rather thick. Since you are manually processing run one sample to test your protocol or 2 if you want to see the difference between the shorter method and the longer. *protocol. .Complete dehydration is dependent on the volume, type of tissue and more importantly, the thickness of the tissue. You can check the tissue before infiltrating with paraffin.to <http://paraffin.to> see if it has cleared, If it appears transparent (like a stained glass window.) proceed with paraffin infiltration. * *Rena Fail*
On Wed, Feb 26, 2014 at 2:02 PM, Tyrone Genade <tgen...@gmail.com> wrote: > Hello, > > I need some input on a manual tissue processing protocol I have received. > > (5 columns: reagents; tiny to large tissues...) > > Reagents\embryoes\small tissue\medium tissue\large tissue > 70% EtOH\30 min--1hr\1 hr--ON\2hr--ON\4hr--ON > 95% EtOH\30 min--1hr\1 hr\1hr\2hr > 95% EtOH\30 min\30 min\1 hr\1 hr > 100% EtOH\30 min\1 hr\30 min\1 hr > 100% EtOH\10 min\30 min\30 min\1 hr > toluene\30 min\1 hr\1hr\1 hr > toluene\none\30 min\30 min\1 hr till clear > parafin infiltration\1hr all > parafin infiltration\1 hr\1 hr\2hr\2hr > EM400\1 hr\1hr\1hr\2+ hr > > I would be replacing the toluene with methylbenzoate. Are there any > anticipated issues? Is this a good protocol so far as producing good > quality tissue for sectioning and analysis? > > I am not sure what is meant by "small" and "large" tissue. My fish are > about 5 cm in length and about 1 cm thick. Would they be classed as small? > > Is it necessary using two different types of paraffin : Surgipath > infiltrating paraffin Cat # 01400 (4-5 lb/bag) and Surgipath embedding > paraffin? For the manual infiltration: would a beaker of paraffin on a > 60oC hotplate and stirrer (on low) be OK? > > I would be fixing with PFA and decalcidying with 30% EDTA. Might refixing > in the PFA after decal be a good idea to make sure the tissue integrity is > maintained? I would probably stored fixed, decalsified samples in 70% EtOH > until I can process fully. Any one think there would be complications from > doing this? > > Thanks > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet