4.6. tdTomato Fluorescence Analysis

Skeletal muscles were fixed in 4% buffered formalin for 24 h and dehydrated with increasing scale of sucrose. Samples were embedded in cryostat-embedding medium (Bio-Optica, Milan, Italy) and frozen. Cryosections of 5 µm were cut with a cryostat (Leica), fixed with 4% paraformaldehyde, dried and rinsed in PBS for 10 min. Slides were stained with DAPI and mounted in Mowiol mounting medium. Microscopy was carried out using an upright Macroscopic-fluorescence microscope (Macroscope, Leica Microsystems SA, Rueil-Malmaison, France), equipped with a Cool Snap HQ Camera (Roper Scientific, Photometrics, Tucson, AZ, USA). Slides were imaged by fluorescence using appropriate filters (TdTomato: excitation filter, BP: 560/40 nm, emission filter 630/75 nm and autofluorescence: excitation filter, BP: 480/40 nm, emission filter, BP 527/30 nm). The files were stored with image acquisition software (Metavue, Metamorph, Molecular Devices, Sunnyvale, CA, USA). The files were stitched with the Adobe Photoshop software to obtain a single image of the whole muscle. Muscle area, mean and integrated fluorescence intensity were calculated with an analysis software (Image J) [54]. After a background correction, by applying a suitable threshold on the TdTomato images, TdTomato positive area, mean and integrated fluorescence intensity were measured in the limit of this threshold. Similarly, the entire muscle area was measured in the limit of a suitable threshold defined on the autofluorescence image.