Histochem Cell Biol (2010) 134:205–213 made from separate regions across each lung section at 400£ magniWcation. Three mice were used from each of the strains at six diVerent postnatal ages for a total of some 162 diVerent photomicrographs (54 per strain). In order to eliminate a possible subjective bias in this study, four objective observers were employed to count the alve- olar wall intercepts. Immunohistochemistry (IHC) IHC staining was done using a BenchMark® XT IHC/ISH staining apparatus (Ventana Medical Systems, Inc, Tucson, AZ, USA), according to the manufacturer’s detection XT iVIEW DAB Open Kit with manual adjustments of the pro- gram in order to accommodate mouse antigens. All anti- bodies were freshly diluted with 2.5% goat serum (Jackson ImmunoResearch Labs, Inc.). TGF 1 antibody (sc-146, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used at 1:200 dilution, prosurfactant protein C polyclonal antibody (pro-spc, 20R-PR030, Fitzgerald Industries Int’l, Concord, MA, USA) for ATII cell labeling at 1:3,000, and biotinylated goat anti-rabbit secondary antibody (Jackson ImmunoResearch) at 1:500. NonspeciWc staining was elimi- nated by pretreatment with 2.5% goat serum and endoge- nous biotin blocked by Blocker A/B reagents provided by Ventana Medical Systems. Slides were counterstained by hematoxylin followed by bluing reagent. Negative controls were treated the same, only without the primary antibodies. Fluorescent dual stainings for surfactant C and -smooth muscle actin ( -SMA) were performed by the core facility in the Departments of Oncology and Pathology at the Johns Hopkins Medical Institutions. BrieXy, the sections were blocked in a mixture of Fab fragment of goat anti-mouse IgG (Jackson ImmunoResearch), protein block solution (Dako Cytomation, Carpinteria, CA, USA), normal goat serum (Fitzgerald Industries Int’l), and bovine serum albu- min (Sigma Chemicals, St. Louis, MO, USA). The primary antibodies used were rabbit polyclonal anti-prosurfactant protein C (Fitzgerald Industries Int’l) and mouse monoclonal anti-smooth muscle actin (Dako Cytomation). Secondary antibodies used were anti-rabbit Alexa-488 and anti-mouse Alexa-610 (Molecular Probes Inc., Eugene, OR, USA). Slides were counterstained by DAPI (Sigma Chemical Co.). ATII cell counting Photomicrographs from prosurfactant protein C-stained slides were analyzed by Image J software for the numbers of ATII cells using the color deconvolution and analyze particles function. The size was set larger than 220 square pixels in order to avoid counting small non-cellular particles. 207 Masson trichrome staining This was done using commercially provided Masson Tri- chrome Stain Kit (Richard-Allan ScientiWc, Kalamazoo, MI, USA) according to the manufacturer’s protocols. ELISA analysis Total TGF 1 levels in BAL supernatants were tested by ELISA assay (Promega, Madison, WI, USA), using acid activated samples according to the manufacturer’s protocol. Each sample was done in duplicate, and results reported as pg TGF 1 per milliliter BAL. Statistical analyses The statistical analyses of strain diVerences at each age and the age related trends of each mouse strain were performed separately by the Tukey HSD test, using JMP software (SAS Institute Inc., Cary, NC, USA). The criterion of sig- niWcant diVerence was set at P < 0.05. Results are reported as averages § standard deviations (§SD). Results Lung Wbrosis in HPS double mutant mice At 12 months of age, large areas of blue Masson trichrome stained collagen were observed in the alveolar epithelium in HPS (Fig. 1a). At 17 months, sections from HPS double mutants had intensely stained blue collagen in association with thickened alveolar walls and tissue patches, the classi- cal histological presentation of lung Wbrosis (Fig. 1b). In contrast, WT and CHS histological samples from all ages tested were devoid of the concentrated blue staining regions, indicative of the lack of collagen deposition in these two mice strains (Fig. 1c, d). At the ages from 2 to 9 months, there was no concentrated blue staining in the lung sections from the three mouse strains (data not shown). The level of lung Wbrosis was quantiWed by hydroxypro- line analyses in the three strains from 2 to over 17 months of age (Fig. 1e). Results indicated that lung Wbrosis in the HPS double mutant at 17 months of age was prominent, which was consistent with the observations from Masson trichrome stained lung sections. HPS lungs also exhibited signiWcantly larger alveoli compared with WT and CHS at all ages tested, indicative of emphysema (Fig. 1f) and consistent with previous studies of this strain (Lyerla et al. 2003). The enlargement of alve- oli became more severe after 12 months of age, when lung Wbrosis was observed. 123