2.10. Whole-mount immunofluorescence staining of IdU/CldU and IdU/EdU

Dissected corneal-limbal tissues were permeabilized with cold methanol at −20 °C for 10 min and rinsed 3 times in PBS. DNA was denatured with 2 M HCL at 37 °C for 15 min, neutralised with 0.1 M sodium borate buffer (pH 8.5) at room temperature for 20 min, followed by rinses with PBS (3 × 10 min) and 2 h incubation in blocking buffer (0.3% BSA in PBS; 4% donkey serum, 0.1% Triton X-100), at room temperature. Tissues were incubated with the primary antibodies, mouse anti-IdU (ab181664) (1:250 dilution) and rat anti-CldU (ab6326) (1:250 dilution) overnight at 4 °C. On the following day, tissues were rinsed with PBS (3 × 20 min), and co-incubated with secondary antibodies, Alexa Fluor 594 goat anti-mouse IgG1 (1:200 dilution) and cross-absorbed goat anti-rat IgG (1:200 dilution) (Millipore-AP136F), for 3 h at room temperature away from light. Tissues were rinsed with PBS (3 × 20 min) and counterstained with TO-PRO-3 Iodide (T3605-Life technologies) (1:1000 dilution in PBS) for 30 min. Following additional rinses with PBS, tissues were cut into 16 sectors to lie flat and mounted on microscope slides using Vectashield mounting medium (H-1000-Vector Laboratories).

The protocol of double-immunofluorescence staining of IdU/EdU was similar to the above IdU/CldU immunofluorescence staining method except for the following: after permeabilization with cold methanol, EdU label was first detected by incubating in Click-iT® EdU reaction -Alexa 488 azide (ThermoFisher {"type":"entrez-nucleotide","attrs":{"text":"C10337","term_id":"1535408","term_text":"C10337"}}C10337) according to manufacturer's instructions. The reaction was carried out in accordance with the manufacturer's instructions, for 30 min at room temperature, protected from light. Then IdU was detected using the same steps described above. There was no cross-reactivity between this IdU antibody and EdU or CldU, shown by injecting mice with EdU or CldU only and then performing anti-IdU immunohistochemistry to confirm absence of signal.