Three 2 mm × 2 mm × 2 mm fragments were cut from three different segments of the right lung and fixed [2.5% glutaraldehyde and phosphate buffer 0.1 M (pH = 7.4)] for electron microscopy analysis (JEOL 1010 Transmission Electron Microscope, Tokyo, Japan).
In each electron microscopy image (50/animal), the following structural changes were analyzed: (a) shedding surface epithelium, (b) airway oedema, (c) eosinophil and neutrophil infiltration, (d) subepithelial fibrosis, (e) smooth muscle hypertrophy, (f) myofibroblast hyperplasia, (g) mucous cell hyperplasia Osimertinib and (i) multinucleated cells (Antunes et al., 2010 and Abreu et al., 2011a). Pathologic findings were graded on a five-point semi-quantitative severity-based scoring system, where 0 = normal lung parenchyma, 1 = changes in 1–25%, 2 = changes in 26–50%, 3 = changes in 51–75%, and 4 = changes in 76–100% of examined tissue. Analysis was performed by two blinded pathologists. Fluorescent images of the basement membrane were obtained using a confocal microscope (Leica Microsystems Ltd., Heidelberg, Germany). Tissue sections were pretreated with PBS for 30 min and incubated overnight at room temperature in a humidified chamber with a mouse antibody against type V collagen (1:50), followed by double staining with fluorescein and rhodamine (rhodamine-conjugated goat SCH772984 anti-mouse IgG-R, dilution 1:40, Santa Cruz Biotechnology, Santa Cruz, CA). For recipients of GFP marrow transplants,
1 week after BMDMC administration, frozen sections were treated
with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI)-supplemented mounting medium Alectinib concentration (Vectashield, Vector Labs, Burlingame, CA), cover-slipped and examined for GFP expression by confocal microscopy. Background autofluorescence was determined through examination of 10 simultaneously prepared negative control sections that were stained only with DAPI. Images were processed and reconstructed using NIH Image software and contrast and colour levels were adjusted in Adobe Photoshop 7.0. The number of GFP+ cells per tissue area was determined by the point-counting technique (Weibel, 1990 and Araujo et al., 2010) across 10 random, non-coincident microscopic fields. Levels of interleukin (IL)-4, IL-13, transforming growth factor (TGF)-β and vascular endothelial growth factor (VEGF) in lung tissue 24 h after the last challenge were evaluated by ELISA using matched antibody pairs from PrepoTech and R&D Systems (Minneapolis, MN, USA), according to manufacturer instructions. Results are expressed in pg/ml. Data were tested for normality using the Kolmogorov–Smirnov test with Lilliefors correction and the homogeneity of variances was assessed with the Levene median test. If both conditions were satisfied, two-way ANOVA, followed by Tukey’s test when required, was used for the comparison of differences among the groups. Nonparametric data were analyzed using ANOVA on ranks followed by Tukey’s test.