Movat pentachrome staining kit

PDGFRA^low/–PDPN⁺CADM1⁺ and PDGFRA^low/–PDPN⁺CADM1^– cells were sorted by flow cytometry and cultured for 7–10 days as previously described.^{97 (link)} Briefly, 5 × 10⁵ cells were resuspended in 5 μL of Matrigel (BD) on ice and then aspirated into a micropipette (Drummond Scientific, 5-000-2010). A small incision was made near the kidney pole to separate the capsule from the renal parenchyma. Matrigel with cells was injected into the kidney pocket. Eight weeks after transplantation, grafts were dissected and fixed in 4% paraformaldehyde at 4 °C for 12 h, decalcified in 10% EDTA at room temperature for 3 days and then dehydrated in 30% sucrose at 4 °C overnight. Grafts were then cryosectioned at 10 μm and stained by Movat Pentachrome Staining Kit (ScyTek, MPS-1) to demonstrate bone and cartilage differentiation. Immunostaining of collagen I and II were also performed on adjacent sections (see below).

Formalin-fixed murine lungs were embedded with paraffin as formalin-fixed paraffin-embedded (FFPE) specimens. FFPE sections were stained using hematoxylin and eosin staining solutions or the Movat Pentachrome Staining Kit (ScyTek, Logan, UT, USA). Image analysis was performed using ImageJ 1.53f. (National Institutes of Health, Bethesda, MD, USA). The binary image was generated from hematoxylin and eosin-stained lung tissue image using auto threshold of ImageJ, and then the MLI was calculated using Measure MLI plugin^{50 (link)}. Distance transform watershed image from the binary image was obtained by Chamfer distance-based watershed using MorphoLibJ^{51 (link)} and Graylevel Watershed (http://bigwww.epfl.ch/sage/soft/watershed/) plugins. The area of each segment in the watershed image was used to calculate the equivalent airspace diameter D₂^{52 (link),53} . The outer circle area (S) and the inner circle area (s) for the bronchial wall were measured using hematoxylin and eosin-stained lung tissue image. Bronchial wall thickness (r) was calculated using the following formula: r = √([S–s]/π).