Pas staining kit

The eyeballs were collected, fixed with 4% paraformaldehyde (PFA), paraffin embedded, sectioned at a 5 μm thickness, and stained with PAS staining kit (Cat#1008, Servicebio, Wuhan, China) according to the manufactory’s instruction. The superior and inferior conjunctiva were examined and photographed with a digital light microscope (Nikon). Three sections from the central parts of the eye from each animal were studied. Six mice were used for each group.

Eyeballs and lacrimal gland were fixed in 4% formaldehyde and embedded in paraffin. The eyeballs tissue was sliced into 4-μM-thick sections, deparaffinized with xylene, and then hydrated in an ethanol gradient. Then, the sections were stained with hematoxylin-eosin staining kit (Servicebio, Wuhan, China). The eyeballs and eyelids collected from mice were fixed in 4% paraformaldehyde, then embedded with paraffin and sectioned into 5 µm thickness. A PAS staining kit (Servicebio, Wuhan, China) was used to stain the sections. For immunohistochemistry analysis, the sections were incubated with anti-Enterovirus A71 antibodies (Abcam, ab36367) at 4°C overnight. Subsequently, the sections were incubated with secondary antibody (Alexa Fluro 488-labeled goat anti-mouse IgG) for 30 min at room temperature. The bound antibody was performed with a DAB substrate kit (Thermo Scientific). Photos were taken of the sections using a light microscope (Leica Microsystems, Wetzlar, Germany).