Masson s trichrome stain

One kidney of each mouse was embedded in paraffin and sliced into 4–5 μm sections [38 (link)]. The sections were stained with hematoxylin and eosin (H&E) (Sigma-Aldrich. Inc., Saint Louis, USA), Masson’s trichrome stain (Sigma-Aldrich. Inc., Saint Louis, USA), and picro sirius red stain (Cis-Biotechnology Co., Ltd., Taichung, Taiwan) according to the manufacturer’s instructions.

Lipoid GmbH, Ludwigshafen, Germany, provided Phospholipon 90 G (95.6 percent Phosphatidylcholine) as a gift sample, which was utilized without additional purification. Liposomes were prepared using HPLC-grade chloroform and methanol. Naringin, D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS), Acetonitrile (HPLC), Cholesterol, Bleomycin, Formaldehyde, Orthophosphoric acid, Potassium chloride, Calcium chloride, Sodium chloride, hematoxylin-eosin (H and E) stain and Biochemistry analysis kits, Masson’s Trichrome stain were purchased from Sigma Aldrich (Bangalore, Karnataka, India) and Cayman Chemical Company (Ann Arbor, MI, USA).

Skin biopsies, obtained from the upper back of patient III:1 and of unrelated age-matched healthy individual and backs and tails of 4 months old mice, were used for histological analysis and for electron microscopy.
Human skin samples and mouse tail and skin samples for histological examination were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH-7.4) overnight at room temperature. Samples were processed for embedding in paraffin and 5 µm-thick sections were stained with hematoxylin-eosin (H&E), or for Masson’s trichrome stain according to manufacturer’s instructions (Sigma-Aldrich). Slides were imaged using an Olympus BX53 upright microscope.
For human samples electron microscopy, skin tissue was fixed with 2.5% glutaraldehyde and 1% paraformaldehyde in Sorenson buffer for 1 h at room temperature (RT), then moved to 4 °C overnight. The tissue was washed with 0.1 M Sodium Cacodylate buffer and stained with 1% Osmium TetraOxide in Cacodylate buffer, and then with 1% uranyl acetate for 1 h at RT. The tissue was then dehydrated and embedded in Epon 812. Ultra-thin (75 nm) sections were cut using Leica UC7 ultramicrotome. Sections were transferred to copper grids and visualized using a Talos L120C Transmission Electron Microscope at an accelerating voltage of 120 kV. Mice samples were visualized using Talos F200C transmission electron microscope operating at 200 kV.

hMSCs (Lonza, Basel, Switzerland) were cultured at 37 °C in Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS). Fibrogenic differentiation was induced by connective tissue growth factor (CTGF) treatment [16 (link)]. Osteogenic differentiation was induced by using a StemPro^® Osteoblast Differentiation Kit (Gibco). Masson’s trichrome stain (Sigma, St. Louis, MO, USA) and Alizarin red (ScienCell Research Laboratories, Carlsbad, CA, USA) were used as previously described [16 (link)].

Small airway fibrosis was measured in inflated lung sections from unchallenged WT and Cc16^−/− mice that were 6, 12, or 18 months of age. Formalin‐fixed paraffin‐embedded lung sections were stained with Masson's Trichrome stain using a commercial kit (Sigma‐Aldrich, St. Louis, MO). Images of both lung fields containing all airways having a diameter of 300–699 μm in each mouse (10–20 airways/mouse) were acquired using microscope interfaced with a digital camera a camera. The thickness of the extracellular matrix protein layer (stained blue) around small airways having a mean diameter of 300–699 microns was measured using MetaMorph software (Molecular Devices, San Jose, CA), as described previously (Laucho‐Contreras et al. 2015b).

Tissues were fixed in 10% (v/v) neutral-buffered formalin, embedded in paraffin, and sectioned. The sections were deparaffinized and stained as previously described^{38 (link)}. Collagen deposition was assessed using Masson’s trichrome stain (Sigma-Aldrich). Haematoxylin and eosin–stained slides were analysed to score the grade of ventricular inflammation. Histological scores were determined as follows: grade 0, no inflammation; grade 1, <10% of the heart section infiltrated; grade 2, 10–30%; grade 3, 30–50%; grade 4, 50–90%; and grade 5, >90%. At least five images per section were acquired for quantification, and the positively stained areas were evaluated using ImageJ software 1.49 v. (http://imagej.net/).