Fast green

Synchondroses in paraffin sections made from the demineralized cranial base were stained with safranin O. Briefly, 4% PFA/PBS-fixed mouse heads were decalcified in 10% EDTA/0.1 M Tris (final pH 7.0) for one week at room temperature with agitation and embedded in paraffin. Paraffin sections (4 μm) were cut with a microtome blade (PATH BLADE + PRO, Matsunami) on a sliding microtome (Yamato Kohki, REM-710). After deparaffinization, sections were stained with Weigert’s iron haematoxylin solution, washed with 0.5% HCl in 70% ethanol, and stained sequentially in 0.05% fast green (Polysciences) and 0.1% safranin O (Polysciences). Slides were then evaluated by microscopy (BX53, Olympus).

The left lung lobes were dehydrated, embedded in paraffin and cut into 5 µm sections and placed on glass slides. The sections were then deparaffinized and stained with 1% toluidine blue (Sigma-Aldrich) followed by counterstain with fast green (Polysciences, Inc., Warrington, PA, USA) to detect mast cells. Mast cells were counted in nine sections per mouse, taken from three cross-sectional places distal to the hilum, each separated by at least 20 µm. To evaluate inflammatory cell infiltration and smooth muscle layer thickness, sections were stained with haematoxylin and eosin (HistoLab). Inflammatory cell infiltration was scored using the following scale: 0 = no inflammatory cell infiltration, 1 = inflammatory cells present centrally, 2 = infiltration centrally and some spread to the parenchyma and 3 = massive infiltration centrally as well as in the parenchyma. The thickness of the smooth muscle cell layer around the central airway was measured and each sample was then given a relative score; 0 = low, 1 = intermediate, and 2 = high ASM score based on the thickness of the ASM layer.

Dulbecco’s minimum essential medium (DMEM), fetal bovine serum, stock antibiotic/antimycotic mixture were obtained from Invitrogen (Carlsbad, CA). ⁴⁵Calcium was obtained from Perkin-Elmer (Boston, MA). Antibody specific to MMP-13 (Lifespan Biosciences, Seattle, WA), ADAMTS5 (Santa Cruz Biotechnology, Dallas, TX), Cox-2 (Santa Cruz Biotechnology, Dallas, TX), and CCL-5 (Bioss, Woburn, MA) were obtained from the commercial sources indicated. Safranin-O, fast green, picrosirius red, and alcian blue were obtained from Polysciences (Warrington, PA). PC and PC-E were prepared according to the method described [32 (link)]. All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO).