Lsm 700 meta microscope

Basal membranes or the central plane of cells were observed by confocal microscopy using a Zeiss LSM700 Meta microscope with a 63× oil immersion lens, and images were acquired and processed using ImageJ (National Institutes of Health). Scale bars, 10 μm.

Immunofluorescence and confocal microscopy imaging were performed as described previously [4 (link)]. The primary antibodies used in our analyses were: FITC-conjugated anti-mouse FoxP3 (FJK-16s, eBioscience, San Diego, CA, USA), APC- conjugated anti-mouse CD3e (145-2C11, eBioscience), and rhodamine-conjugated anti-mouse IgM (Jackson ImmunoResearch, West Grove, PA, USA). Sections were mounted using ProLong Gold Antifade Reagent (Molecular Probes, Carlsbad, CA, USA), and confocal images were obtained using an LSM 700 Meta microscope (Carl Zeiss, Oberkochen, Germany), equipped with 488, 568, and 633 nm lasers. Images were analysed using Zeiss LSM microscopy software Zen 2.6 (Carl Zeiss).

To explore muscle hypertrophy, muscle fiber diameters were measured. The collected muscles, soleus, plantaris and the medial part of the gastrocnemius were cut at mid-belly, fixed in 10% neutral buffered formalin, embedded in paraffin blocks cut at 5 µm thickness and mounted on saline-coated slides as previously described (Kalakh and Mouihate, 2019 (link)). Briefly, muscle sections were incubated with a primary mouse monoclonal antibody anti-myosin type II (1:1000, Sigma-Aldrich) followed by a secondary polyclonal antibody anti-mouse IgG tagged with Alexa fluor 488 (1:400; Thermofisher). Four sections from each muscle were randomly sampled. Images of the cross sections of sampled muscles were acquired using a confocal microscope (Zeiss LSM 700 META microscope). Eight fields at magnification of 20x were randomly selected and images were acquired and analyzed using ImageJ (Schneider et al., 2012 (link)). For each muscle, 250–300 muscle fiber cross sectional areas were measured. For muscle typing, all muscles fibers (immunostained for the fast fibers or unstained) in aquired images were counted and the percentage of fast fibers (Myosin type-II immunoreactive fibers) was calculated.