Anti ck14

For the histological analysis, the mice were sacrificed and tissue samples were obtained on days 3, 6, and 12. The samples were fixed in 4% paraformaldehyde, dehydrated using a graded ethanol series, and embedded in paraffin. The samples were obtained at a thickness of 5 mm and stained with H&E to confirm the presence of the epidermis and cell infiltration. Moreover, staining with Masson’s trichrome (MT) stain was performed to assess the presence of collagen index (thickness and maturation) in the wound-regeneration tissues. To evaluate the immune cell expression (macrophages), re-epithelialization, and proliferation, the tissue sections were stained with anti-F4/80 (Thermo Scientific, Waltham, MA, USA, cat no. 14-4801-82), anti-CK14 (cytokeratin14, Abcam, cat no. ab181595), and anti-Ki67 (Thermo Scientific, Waltham, MA, USA, cat no. 11-5698-82) antibodies. The staining signals were developed using an avidin peroxidase system (ABC kit; Vector Laboratories, Burlingame, CA, USA), and hematoxylin (Sigma-Aldrich, St. Louis, MO, USA) was used for counterstaining.

The esophageal tissues were homogenized in lysis buffer containing proteinase inhibitors (Jianglai Biotech, Shanghai, China)^{53 (link)}. The lysates were centrifuged at 12,000 rpm for 10 min at 4 °C, and the concentrations of the proteins were determined with a protein kit (Bio-Rad Laboratories Inc., Hercules, CA, USA). Twenty micrograms of each esophageal protein was electrophoresed on a 5% sodium dodecyl sulfate-polyacrylamide gel and then transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline including 0.1% Tween-20 for 1 h at room temperature. The membranes were then incubated with primary anti-CD71 (Santa Cruz Biotechnology; 1:200), anti-CK14 (Abcam; 1:100), anti-PCNA (Santa Cruz Biotechnology, 1:100), or anti-integrin α6 (Santa Cruz Biotechnology, 1:100) antibodies overnight at 4 °C and subsequently incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature. Specific protein bands were developed using an enhanced chemiluminescence detection kit (Amersham, Piscataway, NJ, USA). The membranes were probed with β-actin antibody (Abcam), and the intensity of each protein band was normalized to β-actin.

Mouse eyeballs were excised and snap-frozen in Tissue-Tek OCT compound at 2 or 3 days after levobunolol, atenolol, or ICI 118, 551 treatment. The frozen sections (7 μm) were fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 15 min, and then blocked with 5% BSA for 1 h at room temperature. The sections were then incubated with anti-CK3 (Abcam, Cambridge, MA), anti-CK14 (Abcam), anti-CK19 (Proteintech, Chicago, IL), anti-Ki67 (Abcam), anti- phosphorylated epithelial growth factor receptor (pEGFR, Abcam), or anti-phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2, Cell Signaling Technology, Danvers, MA) overnight at 4 °C and subsequently incubated with fluorescein-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h, followed by 4′, 6-diamidino-2-Phenylindole (DAPI, Solarbio, Beijing, China) staining for 5 min. All stainings were observed through an Eclipse TE2000-U microscope (Nikon, Tokyo, Japan) and were quantified by Image J software. Firstly, the images were put on, then, which were conversed to 8 bit and adjusted to gray scale followed by measuring parameters including the area, mean gray value (Mean), integral optical density. Finally, we got average gray scale of these images which were used to statistically analyze.