Axioskop 40 microscope

Sections (4 µm) of each intestinal tissue sample were stained with hematoxylin and eosin for the morphometric study. For this determination, a ZEISS Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany) was used with a Spot Insight camera, and the Spot Advanced software (Spot Imaging Solution, MI, USA). In each slide, the height and crypt depth of 10 villi were measured and the results were expressed in μm. In addition, the villus height/crypt depth ratio was determined. The number of intraepithelial lymphocytes and goblet cells was quantified by counting in 10 fields of epithelium of 25,000 μm².
For immunohistochemical analyses, the detection of IgA-secretory cells in the jejunum and ileum was performed by the avidin–biotin–peroxidase complex technique, according to Oliveira et al. [39 (link)]. In the intestinal lamina propria, the number of IgA-positive cells was checked with a ZEISS Axioskop 40 microscope (Carl Zeiss, Oberkochen, Germany) using a Spot Insight camera and the Spot Advanced software (Spot Imaging Solution, MI, USA). Immunolabeled cells were recorded in 10 non-overlapping consecutive fields of 25,000 μm².
The same researcher, blinded to the treatments, performed the morphometric and immunohistochemical determinations.

Sections of TA muscles were harvested at five or 14 days post-injury from adult or aged Ctl and P7:S4KO mice. Regenerated skeletal muscles harvested five days after injury were immunostained with embryonic Myosin Heavy Chain (eMyHC) antibodies to label actively regenerating fibers and average cross-sectional area of 250–300 myofibers was quantified with ImageJ. Regenerated skeletal muscles harvested 14 days after injury were processed for H and E or Sirius Red staining. For H and E, flash-frozen sections were fixed for 3 min in 4% PFA, stained with Mayers Hematoxylin and Alcoholic Eosin Y, dehydrated, equilibrated with xylene and mounted using Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, MI). For Sirius Red staining, a Picrosirius Red stain kit (Polysciences, Warrington, PA) was utilized. Frozen sections were fixed for 1 hr at 56°C in Bouin’s fixative, washed in water, stained for 1 hr in Picrosirius Red, washed in 1 M HCl, dehydrated, equilibrated and mounted. Bright-field images were collected with a Zeiss Axioskop 40 microscope. To obtain quantification of average cross-sectional area and frequency distribution of 14dpi regenerated fiber size, Myosin/Laminin immunostained TA sections were analyzed using ImageJ software.

C2C12 myotubes were stained with anti-MHC antibody (MF-20, Development Studies Hybridoma Bank at the University of Iowa, Iowa City, IA) followed by FITC-conjugated secondary antibody, and examined using a Zeiss Axioskop 40 microscope coupled to a Zeiss Axiocam MRM camera system that was controlled by Axiovision Release 4.6 imaging software. Acquired images were edited using the Photoshop software. To measure myotube diameter, fluorescein isothiocyanate (FITC)-MHC-stained myotubes were photographed under a fluorescence microscope at ×40. The diameters were measured in a total of 100 myotubes from ≥10 random fields using computerized image analysis (Scion Image, Frederick, MD, USA) as the average at three points along their length. The measurements were conducted in a blind fashion. Cross-sectional area of haemotoxylin and eosin (H&E) stained muscle sections was quantified using the ImageJ software (NIH).