Picrosirius Red Staining Quantification and Visualization of Collagen

Picrosirius red (PSR) staining solution used in staining collagen was made from 0.1% (weight/volume) picrosirius red dye (Sigma-Aldrich) in a saturated picric acid solution. In brief, cells were cultured and irradiated as described above. 48 hours following HF-LED-RL irradiation, cells were fixed in 4% paraformaldehyde at room temperature for 15 minutes, then washed twice with PBS. Cells were then stained with the PSR staining solution for 90 minutes at 37°C. The staining solution was removed and the cells were washed three times with acidified water (0.25% acetic acid). For quantitative analysis, PSR stain was eluted from cells using 200 μL of 0.1 M NaOH, collected in a 96-well microplate, and 540-nm absorbance was read using a Bio-Rad microplate reader.

For light microscopy visualization of collagen staining, cells were cultured on glass coverslips and then stained with picrosirius red solution as described above. Coverslips were mounted with Permount (Electron Microscopy Sciences, USA). The slides were examined and representative photographs were taken at 40× magnification using a BioRevo BZ-9000 microscope (Keyence, Osaka, Japan). Photographs were taken at an exposure of 1/15^th of a second and white-balanced using the BioRevo Image Analysis software.