Fixation of Semi-thin Sections for Combined Light and Electron Microscopy

Abstract

Summary : A mixture of formaldehyde and glutaraldehyde as a fixative in conjunction with GMA-Quetol 523 embedding readily permits the preservation of semi-thin sections for routine use in combined light and electron microscopy. Combined 4% formaldehyde-2% glutaraldehyde fixation gives excellent preservation, and general and histochemical staining performed at the light microscopic level provides satisfactory and GMA-Quetol 523 sections in this range, nuclear and cytoplasmic components such as the nucleoli, mitochondria, Golgi apparatus and cytoplasmic granules, were clarified with various basic and acid dyes, especially hematoxylin and eosin. DNA, glycogen and other polysaccharides were also sufficiently well preserved to studyThe effectiveness of glutaraldehyde, formaldehyde, formaldehyde-glutaraldehyde, periodate-lysine-paraformaldehyde, Bouin's and Zenker's solution on GMA-Quetol 523 embedded tissues was compared. Formaldehyde-glutaraldehyde mixtures promise to be a very useful fixative for use in GMA-Quetol 523 embedded and are suitable for the semi-thin sections necessary in combined light and electron microscopic studies. The results were both visually pleasing and ideal for photomicrography.

Staining of Semithin Tissue Sections Embedded in HPMA, Quetol 523 and MMA

“…This method improved the formulation of GMA, Quetol 523 and MMA by employing HPMA instead of GMA. A low distortion and high stainability were also characteristic of previous GMA-Quetol 523-MMA (Kushida et al, 1981) and HPMA-Quetol 523-MMA embedded tissue sections, but the latter had several advantages over the former for the light microscopy of nerve tissues (Nagato et al, 1989). Quetol 523 and MMA reacted with HPMA and became an integral part of the polymerized system without a cross-linker.…”

“…The application of embedding methods employing water-miscible methacrylates to light and electron microscopy is becoming increasingly well established following the addition of MMA to GMA as a plasticizer (Kushida et al, 1981;Chappard et al, 1983., Liu, 1987). Such semithin sections can yield great detail due to the low distortion and fine resolution.…”

“…In general, the thinner the section the better are the detail and resolution of the image because the structures are less likely to be hidden by constituent overlap within the section thickness (Kushida et al, 1975;Bennett et al, 1976;Nagato et al, 1981;Rieder and Bowser, 1983). Thus, structural details that are not apparent in sections of greater than 2.0 im in thickness may be visible in thinner sections.…”

“…The use of either 2-hydroxyethyl methacrylate (GMA) or 2-hydroxypropyl methacrylate (HPMA) for embedding has been improved by the introduction of Quetol 523 and methyl methacrylate (MMA) (Kushida et al, 1981;Kushida et al, 1985). Quetol 523 is a light-colored, flexible, water-miscible methacrylate (methoxy polyethylene glycol 400 methacrylate).…”

Correlative Light and Electron Microscopy of the Same Sections Embedded in HPMA, Quetol 523 and MMA

“…Small pieces of animal tissues were immersed in a mixture of 4% formaldehyde and 1-2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, as described previously (Nagato et al, 1981). Postfixation with osmium tetroxide should be avoided.…”