For fixed specimens, mineral in caudal fins was stained for 1 h in a 0.1% alizarin red S (AR-S) and 0.8% potassium hydroxide (KOH; both from Sigma-Aldrich) solution and tissues were cleared in 1% KOH for 24 h. Fins were gradually transferred to glycerol for final clearing, preservation and imaging35 . For live specimens, fish were immersed for 15 min in a 0.01% AR-S solution (pH 7.4)37 (link), anaesthetized in MS222, photographed and returned to their containers. Staining was performed in groups of 5 (in 200 mL of staining solution) or individually (in 100 mL of staining solution), according to the need of tracking the same individuals over time.
All fins were photographed under a MZ 7.5 fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a F-View II camera driven by the Cell^F v2.7 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For each fin, bright field and fluorescence images were collected sequentially to assess the regenerated fin and the mineralized area, respectively. Fluorescence micrographs were taken at λex = 530–560 nm and λem = 580 nm with an exposure of 200 ms for fixed specimens and 500 ms for live specimens.
All fins were photographed under a MZ 7.5 fluorescence stereomicroscope (Leica Microsystems GmbH, Wetzlar, Germany) coupled to a F-View II camera driven by the Cell^F v2.7 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany). For each fin, bright field and fluorescence images were collected sequentially to assess the regenerated fin and the mineralized area, respectively. Fluorescence micrographs were taken at λex = 530–560 nm and λem = 580 nm with an exposure of 200 ms for fixed specimens and 500 ms for live specimens.
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