Abstract:
Fixation of biological samples is an important process especially related to histological
and ultrastructural studies. Chemical fixation was the primary method of fixing tissue for
transmission electron microscopy for many years, as it provides adequate preservation of the
morphology of cells and organelles. High pressure freezing (HPF) and freeze substitution (FS) is
a newer alternative method that rapidly freezes non-cryoprotected samples that are then slowly
heated in the FS medium, allowing penetration of the tissue to insure adequate fixation. This
study addresses several issues related to tissue preservation for electron microscopy. Using mice
liver tissue as model the difference between samples fixed chemically or with HPF immediately
after excision, or stored before chemical or HPF fixation were tested with specific focus on the
nuclear membrane. Findings are that immediate HPF is the method of choice compared to chemical
fixation. Of the chemical fixatives, immediate fixation with 2.5% glutaraldehyde (GA)/formaldehyde
(FA) is the best in preserving membrane morphology, 2.5% GA can be used as
alternative for stored and then chemically processed samples, with 10% formalin being suitable
as a storage medium only if followed by HPF fixation. Overall, storage leads to lower ultrastructural
preservation, but HPF with FS can minimize these artifacts relative to other processing
protocols.
