Staining, Histochemistry and Histotechnology
(Frequently Asked Questions)
Dr. John A. Kiernan
Department of Anatomy
and Cell Biology
The University of Western Ontario
Cryoprotection of specimens
Please recommend a way to protect formaldehyde-fixed mouse brains to avoid crack and ice crystal holes that for during rapid freezing. 25% sucrose has been recommended. Should it be in water or phosphate buffered saline?
For ultracryomicrotomy (or should it be cryoultramicrotomy) Tokuyasu (1989) used 2.3 M (= 78%) sucrose in 0.1M phosphate buffer.
He was working with blocks much smaller than mouse brain, so you will no doubt have to increase the time. Inflitration of blocks 1 mm wide usually took 30 minutes. He stated that infusion was complete when the specimen no longer floated on the top of the sucrose solution. The same author reported that 10-30% PVP and 1.6-2M sucrose provided still better postfreezing
conditions (compared with freezing alone).
We presently use 5% PVA (polyvinyl alcohol) in phosphate buffer to cryoprotect bone samples before freezing for enzyme and immunohistochemistry.
One other point that may be worth considering is the method for freezing. If you are thinking of snap-freezing, I would recommend hexane instead of isopentane. Hexane freezes at
a considerably higher temperature: about 80 C. Many moons ago, when I worked in Neuropathology in Scotland, I found that mouse brains tended to crack when frozen in isopentane, but that we had much better preservation when freezing in precooled hexane (we never cryoprotected them though).
Tokuyasu KT. 1989. Use of polyvinylpyrrolidine and polyvinylalchohol cryoultramicrotomy. Histochem. J. 21:163.
It is a common practice to immerse rodent brains in 20-30% sucrose at 4 C, at least until they sink. If they have been fixed for only a short time (less than 48 hours), it is probably best to dissolve the sucrose in PBS rather than water alone.
Rosene et al (1986) found that 20% glycerol with 2% dimethylsulfoxide (DMSO) was better than sucrose. The sucrose concentration needs to be much higher than is commonly used - at leased 60% (see Lepault et al, 1997).
References (with brief notes).
Rosene,DL; Roy,NJ; Davis,BJ (1986): A cryoprotection method that facilitates cutting frozen sections of whole
monkey brains for histological and histochemical
processing without freezing artifact. J. Histochem. Cytochem.
Techniques compared. Optimum cryoprotection with 4 day infiltration (4 C) of 20% glycerol &
2% DMSO in buffer or fixative. Then freeze in isopentane at -75 C (dry ice). Better than other cryoprotectants (sucrose etc) and freezing methods.
Lepault,J; Bigot,D; Studer,D; Erk,I (1997): Freezing of
aqueous specimens: an X-ray diffraction study. J. Microsc. (Oxford) 187(Sep), 158-166.
EM & X-ray diffraction of
freezing of sucrose solutions. Immersion in a
liquid cryogen or high pressure freezing. Sucrose favours formation of amorphous ice; conc
must be 60% or above for freezing in a cryogenic liquid.
John A. Kiernan