Tissue Preparation for Microscopic Analysis

Fresh and post-culture tissue samples were either frozen in Tissue-Tek OCT (optimum cutting temperature; Sakura, Berkshire, UK) medium, using liquid nitrogen-cooled 2-methylbutane (Sigma-Aldrich), or fixed in 4% paraformaldehyde (PFA) for 24 h. PFA fixed tissues were then dehydrated through an ethanol gradient (70, 90, 95, 100%), incubated with two changes of molten paraffin wax, then embedded and allowed to cool. Frozen tissue sections were cut at a thickness of 8 μm using a Leica CM1100 cryostat and fixed for 20 min in − 20 °C cooled methanol before standard staining using Haematoxylin and Eosin (H&E; [20 (link)]). PFA fixed, paraffin embedded (PFPE) tissues were sectioned using a Leica RM2135 microtome (5 μm).