Nis elements viewer 3.20 image analysis software

Histological and immunohistochemical analyses were carried out as previously described [22 (link)]. Briefly, samples were fixed with formalin, and paraffin embedded samples were sectioned. Hematoxylin and eosin (H&E) staining was used in the histological analysis. The purple stain represents the nuclei, whereas the cytoplasm and connective tissue were stained pink or red. The sections were also subjected to immunohistochemical staining using primary and secondary antibodies for immunocytochemical analysis. Briefly, the tissue was treated with antigen retrieval for 20 min and was incubated with 10% goat serum at 37 °C. Next, the tissue was incubated with primary antibodies and left overnight at 4 °C. The sections were counterstained with DAPI to visualize the nucleus. The stained tissue was observed using a Nikon A1_R confocal microscopy system (Nikon), and the images were processed using NIS-Elements Viewer 3.20 image analysis software (Nikon).

Mouse monoclonal antibodies that were specific for CK3 were purchased from Chemicon International (Temecula, CA; Cat. No: CBL218, 1:50), and the p63 antibody was purchased from Abcam (Cambridge, UK; Cat. No: ab3239–500, 1:200). The antibodies cross react with both human and rat antigens. The procedure was conducted according to a published protocol [19 (link)]. Briefly, corneal epithelial cells, uninduced and induced BMuc were fixed with 4% ice-cold paraformaldehyde for 30 min, followed by washing three times with PBS. The plate was incubated with primary antibodies and left overnight at 4 °C. The cells were then incubated with Alexa Fluor 488 rabbit anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC; Chemicon International, Cat. No. AP160 F; 1:200) secondary antibody at 37 °C for 2 h. The cells were counterstained with 4', 6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific, Waltham, MA) and mounted with anti-fading mounting medium (Biorad, Hercules, CA). The stained cells were observed using a Nikon A1_R confocal microscopy system (Nikon, Tokyo, Japan), and the images were processed using NIS-Elements Viewer 3.20 image analysis software (Nikon, Japan).