Polylysin coated glass slides

For immunohistochemistry, excised corneal samples were embedded in frozen section compounds (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 10 µm sections were cut using a HM525 NX cryostat (Thermo Scientific) and collected on polylysin-coated glass slides (Thermo Scientific). Samples were rinsed and blocked in 5% normal goat serum in PBS for 30 min at room temperature. Subsequently, samples were incubated with the primary antibodies at room temperature for 1 hour or at 4 °C overnight. The primary antibodies used were Na⁺/K⁺-ATPase and ZO-1, as well as anti-human nuclei antibodies (Merck Millipore, Massachusetts, USA). Subsequently, samples were labeled with an AlexaFluor 488 conjugated goat anti-mouse IgG secondary antibody (2.5 µg/ml, Life Technology), mounted in Vectashield containing DAPI (Vector Laboratories, California, USA), and visualized using a Zeiss Axioplan 2 fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

For immunohistochemistry, excised corneal sample was embedded in a frozen section compound (Surgipath; Leica Microsystems, Nussloch, Germany), and stored at −80 °C until sectioning. Serial sections of 10 µm sections were cut using a HM525 NX cryostat (Thermo Scientific, Waltham, MA, USA). The sections were collected on polylysin-coated glass slides (Thermo Scientific), and labelled using a mouse IgG1 anti-human nuclei antibody (Table 2) based on the immunocytochemistry approach as described earlier.