Effects of Cold Stress on Tissue Histology

Histological analysis was conducted to investigate the effects of cold exposure on tissue structure. The fish were exposed to cold stress as described above. After cold exposure, the fish were euthanized by immersion into ice-slurry for 3 min as previously described [21 (link)]. The fish were dissected and tissues including brain (cerebellum), gill, heart, kidney, liver, muscle and spleen were collected. The samples were fixed in 4% PFA (Beyotime Biotechnology, Shanghai, China) at 4 °C overnight. After fixation, the samples were dehydrated with alcohol gradients, embedded with paraffin and sectioned into 4 μm slices. The slices were stuck to glass slides, soaked in xylene for dewaxing and rehydrated in ethanol gradients. Finally, the slides were subjected to H&E staining. Photographs of the tissue sections were taken using an Aperio VERSA Brightfield, Fluorescence & FISH Digital Pathology Scanner from Leica (Wetzlar, Germany). Subjective analyses were performed for the photographs to identify changes of tissue structure among the samples from different experimental groups. The photographs were examined independently by different investigators to avoid personal preconceptions when making their judgments.