Muc5ac

Human nasal tissues were stained with PAS, and the PAS-positive cells, which displayed a purple color, were counted via light microscopy (400×) and expressed as the percentage of the total epithelial cells (500 cells counted). IHC was performed as reported elsewhere,13 (link) and the sections were stained using mouse monoclonal antibodies against the following proteins: FoxA2 (1:50; Millipore, Billerica, MA, USA), MUC5AC, and MUC5B (1:100; Santa Cruz Biotech, Santa Cruz, CA, USA). Then, the antibodies were detected via streptavidin-biotin-horseradish peroxidase complex formation. The immunostaining result was considered positive when brown cells appeared following reaction with the reagent 3', 3'-diaminobenzidine. Replacement of primary antibodies with isotype-matched IgG was used as a negative control. The sections were examined via light microscopy (400×), and the patterns of antibody staining were scored in a quantitative manner. The pattern of immunoreactivity was analyzed in the epithelium and subepithelial area. The number of immunoreactive cells (stained brown) was expressed as the percentage of the total cell number (500 cells counted). The immunoreactivity score was assessed by 2 independent observers in a blinded manner, and their results were averaged.

Corneal whole-mount staining was performed as previously described 7 days after epithelial debridement.^{13 (link)} In brief, full-thickness corneal flat mounts were fixed for 1 hour in Zamboni fixative, incubated with 1% bovine serum albumin (Sigma-Aldrich), 2% goat serum, and 0.2% Triton X-100 in PBS for 1 hour to block nonspecific staining. Subsequently, all samples were incubated with a fluorescein isothiocyanate–conjugated mouse anti-β-III tubulin antibody (1:100; Merck Millipore, Darmstadt, Germany) for 24 hours at 4°C. Finally, the flat mounts were examined under an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan). Quantification of corneal innervation was calculated as the percentage of the area positive for β-tubulin staining as previously described.^{14 (link),15 (link)} For corneal section staining, eyeballs were excised and snap-frozen in Tissue-Tek optimum cutting temperature compound (Sakura Finetek, Tokyo, Japan) at 10 days after treatment (n = 4 per group). The sections were stained with MUC-5AC (1:100; Santa Cruz, Dallas, TX), CD44 (1:200; R&D, Minneapolis, MN), ICAM-1 (1:200; Abcam, Cambridge, United Kingdom), VCAM-1 (1:200; Abcam), ZO-1 (1:200; Abcam), and ZO-2 (1:200; Abcam) followed by the respective secondary antibodies. The stainings were examined and photographed with an Eclipse TE2000-U microscope.

Formalin-fixed paraffin-embedded stomach tissues were processed for histology and immunohistochemistry by standard procedures. Primary antibodies used for immunostaining were: GFP (Invitrogen, A11122, 1:200), Ki67 (Abcam, ab16667, 1:100), Mist1 (Santa Cruz, sc-80984, 1:100), Jagged1 (Santa Cruz, sc-6011, 1:100), Notch1 (Cell Signaling, No. 3608, 1:100), Notch2 (DSHB, University of Iowa, C651.6DbHN, 1:200), Notch3 (ProteinTech, 55114-1-AP, 1:100), Notch4 (Millipore, 09-089, 1:100), Cytokeratin 19 (Abcam, ab52625, 1:200), Muc5AC (Santa Cruz, sc-21701, 1:100), Clusterin (Santa Cruz, sc-6420, 1:100), and TFF3 (ProteinTech, 23277-1-AP, 1:100). X-Gal staining was performed as previously described [18] (link).