Histological Analysis of Limbal Stem Cells

LMS (n = 8) from one patient were used for histological analyses at various stages of cultivation (days: 0–2–5–10–14). LMS were washed in phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA), followed by paraffin embedding and sectioning at 7 µm. These sections were cut and mounted on coated slides and dried overnight at 37 ℃. Slides were dewaxed in xylene and hydrated using graded alcohols to tap water. The sections were consequently stained with haematoxylin and eosin (H&E; Sigma) for histological analysis and Sirius Red (Sigma) for detection of fibrillar collagen, and mounted using fluoromount (Southern Biotech). For immunofluorescence, sections were permeabilized with 0.1% Triton X-100 (Sigma), dissolved in 1% BSA in PBS for 10 min and blocked with 10% goat serum in PBS for 60 min. Then, the slides were incubated overnight with primary anti-Tropomyosin (1:250, Sigma T9283) diluted in 0.1% BSA in PBS at 4 °C. Secondary antibody incubation was performed at room temperature for one hour, followed by mounting with VECTASHIELD containing DAPI (Vectorlabs). Images were taken by a blinded investigator using the Zeiss LSM‐870 microscope.