3.6. Freeze Substitution Histological Observation

Prior to analysis, meat samples were kept at −18 °C overnight and then cut into small cubes (5 × 5 × 3 mm³). The unthawed meat cubes were fixed in Carnoy solution (60% ethanol, 30% chloroform, and 10% acetic acid) at −18 °C overnight. After fixation, the samples were dehydrated at 4 °C with a graded series of ethanol solutions (70–90% in 5% increments). Then, the samples were further processed as described in Bao et al. [41] until micrographs were taken by a light microscope (DM 2000, Leica Microsystems CMS GmbH).