115 Dysregulated cross talk between alveolar epithelial cells and stromal cells in IPF reduces epithelial regenerative capacity containing 1% FBS and 1% P/S in the basolateral compartment. At day 7, images of the organoid cultures were taken using a Nikon Eclipse Ti-E microscope (Brightfield; Nikon Instruments Europe, Amsterdam, Netherlands) and the organoid forming efficiency and organoid size (diameter) were quantified manually using Nikon Eclipse Ti software (Nikon Instruments). Immunohistochemistry Cytospin slides were prepared from the unfractionated cell suspension for immunohistochemical analyses. Slides were fixed with acetone (Merck, Darmstadt, Germany) and blocked with 5% bovine serum albumin (BSA) (Sigma-Aldrich) / 1X Phosphate Buffered Saline (PBS) (Gibco) and 0.25% Hydrogen Peroxide (Merck) / 1X PBS, both for 30 minutes at room temperature (RT). The slides were stained overnight with primary antibody solutions for EpCAM (1:1000; Invitrogen, Massachusetts, USA), endothelial cell marker CD31 (1:100; Immunotools, Friesoythe, Germany), stromal cell marker CD90 (1:100; Biolegend, San Diego, CA), or macrophage marker CD68 (1:100; Agilent Dako, Santa Clara, CA) in 1% BSA / 0.1% Triton-X (Merck) / 1X PBS). The next day, slides were washed with 1X PBS and incubated for 1 hour at RT with a secondary antibody solution (1:50 Rabbit-anti Mouse (Agilent Dako) in 1% BSA / 0.1% Triton-X / 1X PBS). After washing the slides with 1X PBS, they were incubated for1 hour at RT with a tertiary antibody solution (1:50 Goat-anti Rabbit (Agilent Dako) in 1% BSA / 0.1% Triton-X / 1X PBS) where after colour was developed using a NovaRed Substrate kit (Vector labs, Newark, CA) according to the company’s manual. Haematoxylin solution (Sigma-Aldrich) was used to counterstain the nuclei. From a total cell count of, at minimum 150 cells, positive stained cells were counted manually. The presence of bronchiolization in IPF lung samples was identified in haematoxylin and eosin (H&E) - stained sections from paraffin embedded lung tissue taken near the site of tissue used for the organoid cultures. Statistics One-way ANOVA with Šídák’s multiple comparisons test was used to assess for statistical differences after testing the normality of the data with Q-Q plots and Shapiro-Wilk test. In case of non-parametric data, the Mann-Whitney test was used for comparisons of two groups and the Kruskal–Wallis test for comparison of more than two groups. P<0.05 was considered statistically significant. 5
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