Ks 400 imaging system

The formalin-fixed paraffin-embedded (FFPE) samples were cut into 3 μm sections, baked at 65°C for 1-2 hours, deparaffinized with xylene and rehydrated with graded ethanol. Antigens were was repaired by high pressure for 2 min. The sections were incubated in 3% H₂O₂ to quench the endogenous peroxidase activity and then incubated with anti-DVL3 (1:100, Abcam, UK) overnight at 4°C. The next day, the sections were incubated with the secondary antibody (ZSGB, China) for 30 min at room temperature, stained with 3,3'-diaminobenzidine (DAB), counterstained with hematoxylin, and finally sealed with neutral balsam. DVL3 was mainly localized in the cytoplasm or cell membrane, and positive expression was determined by the presence of brown granules. The staining signals were quantitatively analyzed with a KS 400 imaging system (Carl Zeiss Vision, Hallbergmoos, Germany). The mean optical density (MOD) of each section was calculated to evaluate the staining intensity of DVL3.

For each species, the number of fibers and the area occupied by each type of muscle fibers was quantified by morphometric analysis at ×200 magnification. The procedure was based on a hardware system consisting of a Zeiss Axioplan microscope equipped with a ProgRes C10 plus color camera (Jenoptik, Germany) connected to an IBM‐compatible PC, and using software designed for morphometry and color analysis (KS 400 Imaging system, Carl Zeiss Vision GmbH, München, Germany). This image‐analyzing system discriminated between immunoreactive muscle fibers based on differences in color and contrast. For each image, eight standardized microscopic fields of 97.2 μm² each were selected at random separately in the dorsal and ventral muscular layers. For each lizard (n = 3 per species), the absolute and relative areas occupied by the three categories of muscle fibers were calculated in the ventral and dorsal parts, as well as in the body and tail parts. Each measure was performed in triplicate on three consecutive histological slides. For each type of muscle fiber, the area of the fibers was divided by the total number of fibers to estimate the mean individual area of each type of fiber in cross sectional view in the dorsal and ventral parts (and also in the body and tail areas).

For each culture, the number of neurones, the length of neuronal processes and the area occupied by astrocytes were quantified by morphometric analysis at 100× magnification. The procedure utilized a software designed for morphometry and colour analysis (KS 400 Imaging system, Carl Zeiss Vision GmbH, München, Germany). For each culture condition, 5 microscopic fields were picked at random representing a total scanned surface of 1.8 mm². The number of neurones and the mean length of neuronal processes were quantified after MAP2 immunostaining. The surface occupied by astrocytes was calculated on cultures exposed to anti-GFAP immunofluorescence. For each time point, measures were done on 4 independent cultures and results were presented under box plots. Results obtained from morphometric analysis were submitted to non-parametric Mann-Whitney test (the limit of significance set at p < 0.05 by comparison to control values).