Simple method of thawing cryo-stored samples preserves ultrastructural features in electron microscopy

AbstractPreservation 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 insufficient ly 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 invest igated 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 wit h 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...
Source: Histochemistry and Cell Biology - Category: Biomedical Science Source Type: research