[Histonet] manual tissue processing protocol

[Tyrone Genade]

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