Application suite las microscope software

The prostate tissues were fixed in 10% formaldehyde, dehydrated, and embedded in paraffin. Paraffin was sectioned at 4 μm using a microtome (Leica, Werzlar, Germany). For H&E staining, the sections were stained in hematoxylin for 5 min, and then washed with water for 5 min. Then, the sections were stained in eosin for 30 s, dehydrated, and mounted using routine methods. The slides were examined using the Leica DM6 Research Inverted Phase microscope (Leica, Werzlar, Germany). Epithelial thickness and lumen area were measured using Leica Application Suite (LAS) microscope software.

All murine ESC lines were differentiated as previously described62 (link). To initiate embryoid body (EB) formation, “hanging drops” composed of 2000 cells in 30 μL of differentiation medium were generated (day-0 of differentiation). The differentiation medium was based on Iscove’s modified Dulbecco’s medium (IMDM, Gibco) and supplemented with 20% heat inactivated FBS, 0.5 mM monothioglycerol, lacking supplemental LIF. On day-2 of differentiation, the EBs were transferred into gelatin coated 24-well plates with 1–2 EBs per well and cultivated for 2 additional days. From day 5 until day 12, differentiation medium was replaced every 2–3 days. Images of EBs were captured at×5 magnification with a Leica DMIRB inverted phase contrast fluorescence microscope with a DFC425 camera (Leica) and processed using the Leica Application Suite (LAS) microscope software. The microscopic images of the fluorescent EBs were further analyzed for the quantitative analysis of total fluorescence in each EB. Total fluorescence per EB (TF/EB) was calculated in an excel sheet by applying the measurements obtained from the EBs using Image J software. Total fluorescence per EB (TF/EB) = Integrated Density - (Area of selected EB × Mean fluorescence of background readings).

The left side of the mandibles were fixed in 10% neutral buffered formalin for 18 h and demineralized in a solution of 0.1 M ethylene diamine tetraacetic acid for 2 months. After dehydration with ethanol and xylene, the specimens were embedded in paraffin. Serial sections of 7 μm thickness were obtained and stained with hematoxylin and eosin (H&E). The digital images were obtained from Leica Application Suite (LAS) microscope software (Leica Microsystems, Buffalo Grove, IL, USA) with the ×40 magnification.

Skin tissues from the upper back of mice were fixed in 4% formalin for 24 h, dehydrated. After embedding of each specimen, the specimens were sliced to 4 μm of thickness. Sections of skin specimens were stained with hematoxylin and eosin (H&E). Stained specimens were cover-slipped with VECTA Mount permanent mounting medium (VECTOR laboratories, Inc., Burlingame, CA). Digital images were obtained from Leica Application Suite (LAS) Microscope Software (Leica Microsystems Inc., IL, USA). The magnification of H&E staining was ×100.

On day 49, the mice were sacrificed under anesthesia with the injection of avertin (Sigma-Aldrich Inc., St. Louis, MO, USA) and lung tissues were dissected and immersed in 10% neutralized formalin for 24 h. Following tissue fixation, lung tissues were washed and dehydrated by the series concentration gradient of ethanol and xylene for embedding in a paraffin block. The blocks were sectioned at 4 μm thickness and stained by hematoxylin and eosin (H&E), periodic acid–Schiff (PAS) and Masson’s trichrome solution. Tissue slides were visualized with Leica Application Suite (LAS) Microscope Software (Leica Microsystems Inc., Wetziar, Land Hessen, Germany) to determine the histological structure, collagen deposition and reaction of mucin.