Fig. 4

Quantification of inflammatory (IL-1β) and fibrotic (TGF-β) transcripts by RNAScope (Fluorescent in situ hybridization-based RNAScope assay). A Original (left) and magnified (right) images of bleomycin-challenged mouse lung tissue. RNAScope was used to detect TGF-β (green dots, mask outlined in orange) and IL-1β (red dots, mask outlined in turquoise) mRNA. Nuclei (blue, mask outlined in yellow) were stained with DAPI. Right panel shows magnified section of left image. ZenBlue 3.1 (Zeiss) software was used to generate masks to differentiate between true RNAScope signal and autofluorescence or background signal. Autofluorescence and background signal were excluded from the quantification of transcripts and nuclei. Size bar = 50 μm, left; 10 μm, right). B Quantification of transcripts of fibrosis (TGFβ) and inflammation (IL-1β) by RNAScope. Counts were adjusted for cell number as detected by DAPI nuclear count within the same image area. C Difference between the quantity of inflammatory (IL-1β) and fibrotic (TGFβ) markers between the mice challenged with 60 U/Kg bleomycin and age-matched controls as detected by RNAScope. Reported data display average of 10–13 images for each bleomycin dose and timepoint combination. Significance markers depict a difference with p < 0.01 for ANOVA followed by Tukey’s Post-Hoc Test