A NEW METHOD FOR FLAT-EMBEDDING LARGE NATIVE CRYOSTAT SECTIONS FOR TARGETING SMALL PRENEOPLASTIC LESIONS IN COMPARATIVE ULTRASTRUCTURAL ANDULTRACYTOCHEMICAL INVESTIGATIONS
C. Metzger et al., A NEW METHOD FOR FLAT-EMBEDDING LARGE NATIVE CRYOSTAT SECTIONS FOR TARGETING SMALL PRENEOPLASTIC LESIONS IN COMPARATIVE ULTRASTRUCTURAL ANDULTRACYTOCHEMICAL INVESTIGATIONS, HISTOCHEM C, 110(3), 1998, pp. 323-332
Ultrastructural studies of rare and small cellular lesions in patholog
ically altered tissue are difficult to perform, by applying convention
al electron microscopic preparation. The search for lesions, often con
sisting of only a few cells in randomly obtained small specimen blocks
, is time consuming and often without success. The methodological requ
irements for comparative enzyme cytochemical and morphological studies
, i.e., preservation of both enzyme activity and ultrastructure, are d
ivergent. By processing large native cryostat sections for electron mi
croscopy, small preneoplastic focal lesions were successfully targeted
ir liver and kidney. Glucose-6-phosphatase,; alkaline phosphatase, ac
id phosphatase, catalase, and cytochrome c oxidase activities were dis
tinctly localized to endoplasmic reticulum, canalicular membrane, lyso
somes, peroxisomes, and mitochondria, respectively, in the morphologic
ally altered cells. Fixation of serial cryostat sections and enzyme re
actions were both carried out through a semipermeable membrane except
those for cytochrome c oxidase, which was demonstrated after fixation
through the membrane by floating the section in incubation medium cont
aining cytochrome c. Thereafter, the sections were flat embedded and p
olymerized between epoxy resin disks and aluminum dishes fitting exact
ly together. The objects of interest were identified in the light micr
oscope, cut out, and reembedded in reversed gelatine capsules. By usin
g this technique an ultrastructural preservation was achieved similar
to that seen after immersion fixation. The enzyme activities were clea
rly localized without diffusion of the reaction product or unspecific
deposits. The procedure permits precise targeting and complex studies
of rare and small lesions, and opens new perspectives for the use of c
ryo-preserved tissue.