Halo v3

Tissues were fixed in 10% neutral-buffered formalin for 18 hr, embedded in paraffin after graded-ethanol dehydration, and sectioned into 6 μm sections using a microtome. Automated staining was carried out on the BondRxm (Leica Biosystems). Following dewaxing and citrate-buffered antigen retrieval, sections were stained with the primary antibody for 1 hr at RT. Sequential chromogenic detection was performed with the Bond IntenseR (Rat primary) and Bond Polymer Refine Red (Rabbit primary) detection kits (Leica Biosystems). The primary antibodies used were rat anti-CD45 (Invitrogen clone 30-F11, #14-0451-82) followed by rabbit anti-RFP/TdT (Rockland, # 600-401-379). Stained sections were dehydrated in graduated ethanol and xylene washes then mounted with xylene-based Cytoseal (Thermo Fisher).
Stained slides were imaged at 20X magnification with the Zeiss Axioscan slide scanner. For image analysis, Halo v3 (Indica Labs) was used to deconvolve dual stained images into single channels and pseudo colored with blue representing nuclei (hematoxylin), green representing CD45+ cells (DAB stained), and red representing Osx+ cells (RFP stained).

IHC images were analyzed in HALO v3.1 (Indica Labs). For all analyses, images were annotated manually to exclude out of focus and damaged areas, and a random forest classifier was applied to intact areas of the sections to detect epithelial areas. All following analyses were done in epithelial areas only. For goblet cell counting, algorithm CytoNuclear 2.0.9 was used to segment cells based on nuclear staining and categorize them as AB/PAS (mucin) positive and negative cells based on cytoplasmic AB staining. Goblet cell counting was represented as the percentage of AB positive cells of total cells.
To measure epithelial thickness, the apical and basal sides of the epithelium in the sections were manually delineated using a pen annotation tool. Distance between paired apical and basal annotations was measured every 10 μm along the epithelium, and recorded in sequence as epithelial thickness.
To measure intraepithelial and intercellular AB staining, algorithm Area Quantification v2.1.7 was used. Two thresholds were applied to capture AB staining: a lower one to detect all AB staining in both goblet cells and intercellular areas, and a higher one to detect the stronger AB staining inside goblet cells only. Intercellular AB area was calculated by subtracting goblet AB staining (higher threshold) from total AB staining (by lower threshold).