Warp red chromogen

The RNAscope platform (ACD Bio, Newark, CA) was used to quantify gene transcripts in situ. Probes against two of the most significant genes in rejection, and with readily available commercial assays, namely CXCL9 (cat # 440161) and CXCL10 (cat # 311851) (ACD Bio, Newark, CA) were applied on consecutive 2 μm-thick FFPE tissue sections and were detected by alkaline-phosphatase-based technique (RNAscope 2.5 HD Assay—Red) coupled with Warp Red chromogen (Biocare Medical, Pacheco, CA) followed by hematoxylin counterstain. Depending on the abundance and distribution of the transcript, the positive signal can be seen as separate dots or fused group(s) of multiple dots. After detection, the tissue sections were digitized by Aperio ScanScope XT. Whole-slide digital images were analyzed by the Definiens Tissue Studio platform's Dot Count module. In brief, the software models an “average” cell based on the hematoxylin counterstain first, and assigns each dot to a particular cell and counts them. Thresholds were adjusted individually and accuracy of the settings was checked by evaluation of 12 randomly selected HPFs at 40X. The number of dots in the entire section/1,000 cell characterized the expression level of a given gene.

Human airway tissue from asthmatic and non-asthmatic donors and ALI cultures grown from non-asthmatic AEC (untreated and IL-13 treated) were fixed in 10% formalin, embedded in paraffin, and sectioned into 4 μm slices. Sections were deparaffinized in CitriSolv (Fisher Scientific, Toronto, ON, 22-143-975) and rehydrated. Antigen retrieval was performed by autoclaving in Citra solution at pH 6.0 (Life Technologies, Burlington, ON) for 22 minutes. All sections were blocked consecutively using Background Sniper (Biocare Medical, Markham, ON, B5966L) and Dual Endogenous Block (Dako, Burlington, ON, K5361). Sections were stained with anti-SP-D (0.25 μg/mL), MUC5AC (1 μg/mL), or CK-5 (1 μg/mL) antibodies using biotin-free MACH 3 AP-Polymer Detection kit (Biocare Medical, Markham, ON, M3U532 for mouse and M3R533 for rabbit primary antibodies) containing alkaline phosphatase with Warp Red Chromogen (Biocare Medical, Markham, ON, WR806) as substrate. Matched isotype control antibodies were used as negative controls for IHC to demonstrate antibody specificity. Hematoxylin was used for counter-staining of the nuclei. Colour segmentation via ImagePro Plus (Media Cybernetics, Silver Spring, MD) was performed for quantification whereby area positive was normalized to total epithelial area.