Direct red 80 kit

10μM sections were stained with Pico Sirius Red using the Direct Red-80 kit (Sigma) and assessed for collagen using light microscopy. Collagen staining (Red) was quantified using ImageJ.

Lenticules were formalin-fixed and processed for paraffin embedding. Sections (4 μm thick) were stained with H&E and photographed using 10x objective (AxioImager Z1). Thickness at 5 standard positions (¹/₁₀, ³/₁₀, ⁵/₁₀, ⁷/₁₀ and ⁹/₁₀) along the lenticule length was measured using Image J software (NIH) and the mean thickness was calculated. Sections were stained with Picrosirius red (Direct Red 80 kit, Sigma) for global collagens, Periodic acid-Schiff kit (Merck) for global glycoproteins and Alcian blue (Sigma) for global glycosaminoglycans. Under light microscopy, images were taken at 5 standard locations (same as above) under same exposure time, incident light intensity, gamma level and white balance. Staining intensity was quantified using Image J, adjusted with background threshold and calculated as intensity per unit area of lenticule. Results were normalized with the signal intensity of untreated lenticules and expressed as the percentage changes. Of note, lenticules at similar stromal depth were examined in the same round of H&E and three histochemical stainings.