Leica icc50 digital camera

The EpiDerm™ tissues were pre-fixed overnight at 4 °C in 10% paraformaldehyde. Alcohol gradient dehydration for histological characterization was performed by sequential treatment of 3 samples per group with 30% EtOH for 2 h, 50% EtOH for 2 h, 70% EtOH overnight, 95% EtOH twice for 3 h, and 100% EtOH twice for 1 h each time. Tissue samples were then sectioned and stained using standard HE staining.
The slides were examined and recorded using 20× and 40× magnifications with a Leica DM750 microscope, a Leica ICC50 digital camera, and cellSens microscope imaging software (Olympus Corporation, Warsaw, Poland).
The thickness of the corneal layer and the total EpiDerm™ thickness were defined as indicators of the appropriate activity and function of the model after treatment [19 (link)]. All measurements were performed in randomly selected fields of view at 20× magnification (12 photos per group). For histological examination, 40× magnification was used.

Frozen sections were air-dried at room temperature for 15 min and transferred to a freshly prepared solution containing 50 mM Tris-HCl (pH 9.5), 0.14 mg/mL Nitro Blue Tetrazolium (Sigma Aldrich, Milano, Italy), 0.07 mg/mL 5-bromo-4-chloro-3-indolylphosphate (BCIP, Sigma Aldrich, Milano, Italy), and 2.3 mM Magnesium Chloride. Sections were extensively washed in water and counterstained in 0.5% eosin, washed in water, dehydrated with 95% and absolute ethanol, cleared in BioClear clearing reagent (BioOptica, Milano, Italy), and mounted with a resinous medium. Images were acquired with a Leica DM500 microscope with a 20× objective equipped with a Leica ICC50 digital camera (Leica, Wetzlar, Germany), using identical parameters. The full section was reconstructed by stitching the images with the Trak2EM function in Fiji software. As previously described [30 (link),32 (link),33 (link)], quantitative analysis of capillary density was determined by counting the alkaline phosphatase-positive dots in five 1000 px² fields of each muscle section, and the number of capillaries per fiber was determined by counting alkaline phosphatase positive dots surrounding 50 randomly chosen fibers.

SDH staining has been performed as previously described [30 (link),32 (link)]. Briefly, frozen sections were air-dried at room temperature for 15 min, transferred to a freshly prepared SDH solution containing Tris-HCl 0.2 M (pH 7.4), Natrium Succinate 0.2 M and Nitro Blue Tetrazolium (Sigma Aldrich, Milano, Italy) 1 mg/mL, and incubated 15–20 min at 37 °C. The enzymatic reaction was stopped by dipping the slides in water and subsequently fixing them for 10 min in 4% paraformaldehyde. Specimens were extensively washed and mounted in gelatin–glycerol. Images were acquired with a Leica DM500 microscope with a 20× objective, equipped with a Leica ICC50 digital camera (Leica, Wetzlar, Germany), using constant acquisition parameters. The full section was reconstructed by stitching the images with the Trak2EM function in Fiji software and converted to grayscale to measure the intensity of SDH staining. The intensity of staining was evaluated by measuring the mean gray intensity of five 1000 px² fields in each tissue section and in a fiber type specific by carefully drawing a polygon around the perimeter of 50 MyHC I and II myofibers.