Lsm 700 meta laser microscope

Time lapse imaging microscopy was performed on the Leica DMI6000 system. The × 10 images were captured every hour. The green and red fluorescence images were merged and compiled to short videos with the Volocity Demo software. An Axioplan2 microscope (Carl Zeiss) with the Openlab 3.1.7 software (Improvision) was used for fluorescence microscopy analyses. An LSM 700 Meta laser microscope with the LSM 6.0 Image Browser software (Carl Zeiss) was used for confocal analyses. Representative images were selected from at least two independent experiments. For longitudinal sections, 1 in every 20 sections was selected and counted. For transverse sections, 1 in every 15 sections was selected and counted. Blinded counting was performed, as the operator counting the cells was not aware of the type of sample. For counting dedifferentiation frequency, the right (XIAP) and left (control) forelimbs were collected from seven animals at 12 d.p.a. and sectioned transversely. All YFP⁺ nuclei were counted from the regenerate tip to stump until no more YFP⁺ nuclei were found. The number of YFP⁺/MHC⁻ cells in the blastema was normalized to the labelled YFP⁺ myonuclei in the stump (YFP⁺/MHC⁺) before comparing to control.

Frozen sections (5-10 μm) were thawed at room temperature and fixed in 4% formaldehyde for 5 min. Sections were blocked with 5% donkey serum and 0.1% Triton-X for 30 min at room temperature. Sections were incubated with anti-GFP (Abcam 6673), anti-PAX7 (DSHB) or anti-MHC (DSHB) overnight at 4°C and with secondary antibodies for 1 hour at room temperature. Antibodies were diluted in blocking buffer and sections were mounted in mounting medium (DakoCytomation) containing 5 μg/ml DAPI (Sigma). EdU detection was performed as previously described^{15 (link)}. An LSM 700 Meta laser microscope with LSM 6.0 Image Browser software (Carl Zeiss) was used for confocal analyses. One in every 8 sections was selected and labelled. For PAX7⁺ satellite cell counting, 3 sections were randomly selected and counted. For blastema YFP⁺ cell counting, all the sections in the region from regenerate tip to the bone were counted.