The effects of slow freezing and thawing on enzyme compartmentalizatio
n and ultrastructure were studied in rat liver slices frozen in dry ic
e, isopentane/ethanol-dry ice, or liquid nitrogen, and stored at -80 d
egrees C for 1-14 days. Non-frozen slices served as controls. Frozen l
iver slices were thawed in a Karnovsky fixative and processed for tran
smission electron microscopy (TEM). After all freezing protocols, the
outer zone of frozen-thawed tissue was ultrastructurally very similar
to that of non-frozen liver. Towards the center of the tissue, the ult
rastructure progressively deteriorated. Comparison with 50-mu m cryost
at sections prepared for TEM showed that thawing and not freezing is t
he detrimental step for fair preservation of ultrastructure. After tha
wing, homogenization, and differential centrifugation, distribution pa
tterns of soluble marker enzymes were analyzed (cytosol, lactate dehyd
rogenase; mitochondrial matrix, glutamate dehydrogenase; lysosomes, ac
id phosphatase). The enzyme activities were not affected by storage fo
r 2 weeks and the activity distributions showed that protein leakage f
rom compartments was only minimally increased in frozen-thawed tissue
compared with that from non-frozen tissue, irrespective of the method
of freezing. In conclusion, fairly large tissue slices (20 x 5 x 3 mm)
may be frozen and stored at -80 degrees C for biochemical, ultrahisto
chemical or ultrastructural studies. For ultrastructural analysis, onl
y the periphery of the tissue slice should be used.