Metamorph offline version 7.7.0.0 image analysis software

Finally, the SEs were fixed with Carnoy's fixative and embedded in paraffin. To stain for type IV collagen, antigen retrieval was performed by using proteinase K (Roche Applied Science, Penzberg, Germany). For other antibodies, thermal antigen retrieval was performed by using Trilogy solution (Cell Marque) and a pressure cooker. Immunohistochemical staining was performed by using the avidin-biotin-peroxidase complex technique (DAKO). All experiments were repeated twice. For image analysis, same condition was applied for staining and taking pictures. After taking pictures, expression of type IV collagen and integrins was measured by stained areas in three representative areas. In case of PCNA (proliferating cell nuclear antigen) and p63, expression was measured by OD (optical density: intensity/stained area) in three representative areas. Images were analyzed quantitatively using MetaMorph Offline version 7.7.0.0 image analysis software (Molecular Devices, Downingtown, PA). To see the effects of phlorizin on fibroblasts, SEs were stained with Hoechst 33342 (1 μg/mL, 40047, Biotium Inc., Hayward, CA, USA) for 10 min at RT. Then number of dermal cells in 3 representative areas was compared.

For histopathological examination, the lung tissues were stained as described previously [19 (link)]. The 4 % paraformaldehyde-fixed tissues were embedded in paraffin, sectioned at 4 μm thickness, and stained with hematoxylin and eosin (H&E) stain solution (hematoxylin; Sigma MHS-16 and eosin; Sigma HT110-1-32) and periodic acid − Schiff (PAS) (IMEB Inc., San Marcos, CA) to measure inflammatory cell accumulation and mucus production, respectively. Quantitative analysis for airway inflammation and mucus secretion was evaluated using a MetaMorph Offline version 7.7.0.0 image analysis software (Molecular Devices Inc., Sunnyvale, CA, USA).