On the long-term storage of tissue for fluorescence and electron microscopy: lessons learned from rat liver samples.

Occasionally, tissue samples cannot be processed completely and are stored under varying conditions for extended periods. This is particularly beneficial in interinstitutional studies where a given research setting may lack the expertise or infrastructure for sample processing, imaging and data analysis. Currently, there is limited literature available on the controlled storage of biological tissues in primary fixatives for fluorescence and electron microscopy. In this contribution, we mimicked various tissue storage scenarios by taking different fixation conditions, storage temperatures and storage durations into account. Rat liver tissue was used for its well-known diversity of cellular ultrastructure and microscopy analysis. Fluorescent labelling of actin, DNA and lipids were employed in conjunction with high-resolution electron microscopy imaging. Herein, we tested three different fixative solutions (1.5% glutaraldehyde, 0.4% glutaraldehyde and 4% formaldehyde and 4% formaldehyde) and stored samples for 1-28 days at room temperature and refrigerator temperature. We found that liver tissue can be stored for up to 2 weeks in a 0.4% glutaraldehyde + 4% formaldehyde fixative solution, while still enabling reliable fluorescent labelling and ultrastructural studies. Ultrastructural integrity was eminent up to 1 month using either glutaraldehyde or formaldehyde fixation protocols. When liver tissue is fixed with a mixture of 0.4% glutaraldehyde and 4% formaldehyde and stored at 4 °C, it retains its capacity for electron microscopy analysis for several years, but loses its capacity for reliable fluorescent labelling studies. In conclusion, we demonstrated that liver tissue can be stored for extended periods enabling profound structure-function analysis across length scales.