Rapid combined light and electron microscopy on large frozen biological samples

Abstract

SummaryThe use of large unfixed frozen tissue samples (10 × 10 × 5 mm 3 ) for combined light microscopy (LM) and electron microscopy (EM) is described. First, cryostat sections are applied for various LM histochemical approaches including in situ hybridization, immunohistochemistry and metabolic mapping (enzyme histochemistry). When EM inspection is needed, the tissue blocks that were used for cryostat sectioning and are stored at −80 • C, are then fixed at 4 • C with glutaraldehyde/paraformaldehyde and prepared for EM according to standard procedures. Ultrastructurally, most morphological aspects of normal and pathological tissue are retained whereas cryostat sectioning at −25 • C does not have serious damaging effects on the ultrastructure. This approach allows simple and rapid combined LM and EM of relatively large tissue specimens with acceptable ultrastructure. Its use is demonstrated with the elucidation of transdifferentiated mouse stromal elements in human pancreatic adenocarcinoma explants grown subcutaneously in nude mice. Combined LM and EM analysis revealed that these elements resemble cartilage showing enchondral mineralization and aberrant muscle fibres with characteristics of skeletal muscle cells.

Routinely frozen biopsies of human skeletal muscle are suitable for morphological and immunocytochemical analyses at transmission electron microscopy

“…10 However, scarce information exists on the possibility to use frozen biopsies of skeletal muscle for EM 11 and in particular for ultrastructural immunocytochemistry.…”

Imaging Enzymes at Work: Metabolic Mapping by Enzyme Histochemistry

“…These protection measures keep both the activity of the enzymes and the morphology of the tissue in the section unaffected, in such a way that even electron microscopy can be performed after incubation (Figure 11). Frozen tissue in general has a morphology that can be used for electron microscopy (Vogels et al 1993(Vogels et al ,2009, despite the fact that the general opinion is that this is impossible because of ice crystal formation during freezing. Even cryostat sections after enzyme incubation under tissue-protecting conditions show acceptable morphology.…”

Differential Activity of NADPH-Producing Dehydrogenases Renders Rodents Unsuitable Models to Study IDH1R132 Mutation Effects in Human Glioblastoma

“…Samples of tissues were slowly snap-frozen in small plastic vials in liquid nitrogen and stored at -80C until used as described by Vogels et al (2009) to ensure preservation of optimum tissue morphology. Cerebrum, cerebellum, spinal cord, tongue, small and large intestines, pancreas, liver, and kidney were collected.…”