Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Galhuber, M; Kupper, N; Dohr, G; Gauster, M; Kwapiszewska, G; Olschewski, A; Jandl, K; Gschwandtner, E; Schweiger, M; Kratky, D; Leitinger, G; Prokesch, A; Kolb, D.
Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy.
Histochem Cell Biol. 2021; 155(5):593-603
Doi: 10.1007/s00418-020-01952-z
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- Führende Autor*innen der Med Uni Graz
- Galhuber Markus
- Kolb Dagmar
- Prokesch Andreas
- Co-Autor*innen der Med Uni Graz
- Dohr Gottfried
- Gauster Martin
- Gschwandtner Elisabeth
- Jandl Katharina
- Kratky Dagmar
- Kupper Nadja Julia
- Kwapiszewska-Marsh Grazyna
- Leitinger Gerd
- Olschewski Andrea
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- Abstract:
- Preservation of ultrastructural features in biological samples for electron microscopy (EM) is a challenging task that is routinely accomplished through chemical fixation or high-pressure freezing coupled to automated freeze substitution (AFS) using specialized devices. However, samples from clinical (e.g. "biobanking" of bulk biopsies) and preclinical (e.g. whole mouse tissues) specimens are often not specifically prepared for ultrastructural analyses but simply immersed in liquid nitrogen before long-term cryo-storage. We demonstrate that ultrastructural features of such samples are insufficiently conserved using AFS and developed a simple, rapid, and effective method for thawing that does not require specific instrumentation. This procedure consists of dry ice-cooled pre-trimming of frozen tissue and aldehyde fixation for 3 h at 37 °C followed by standard embedding steps. Herein investigated tissues comprised human term placentae, clinical lung samples, as well as mouse tissues of different composition (brown adipose tissue, white adipose tissue, cardiac muscle, skeletal muscle, liver). For all these tissues, we compared electron micrographs prepared from cryo-stored material with our method to images derived from directly prepared fresh tissues with standard chemical fixation. Our protocol yielded highly conserved ultrastructural features and tissue-specific details, largely matching the quality of fresh tissue samples. Furthermore, morphometric analysis of lipid droplets and mitochondria in livers of fasted mice demonstrated that statistically valid quantifications can be derived from samples prepared with our method. Overall, we provide a simple and effective protocol for accurate ultrastructural and morphometric analyses of cryo-stored bulk tissue samples.
- Find related publications in this database (using NLM MeSH Indexing)
- Animals - administration & dosage
- Cryopreservation - administration & dosage
- Freezing - administration & dosage
- Lipid Droplets - ultrastructure
- Liver - ultrastructure
- Mice - administration & dosage
- Mice, Inbred C57BL - administration & dosage
- Microscopy, Electron - administration & dosage
- Mitochondria - ultrastructure
- Find related publications in this database (Keywords)
- TEM
- Cryo-storage
- Cryoprotectant-free
- Ultrastructural features
- Sample preparation
- Biobanking