Safranin o fast green counterstaining

Safranin O/Fast green staining was used to assess glycosaminoglycans (GAGs) content. Samples were fixed in 10% buffered formalin phosphate solution (Fisher Chemical, Fair Lawn, NJ, United States) overnight and dehydrated with an increasing ethanol gradient. After being soaked in xylene for 2 h and then in liquid paraffin overnight, samples were embedded in paraffin. The blocks were sectioned at 6 μm thickness using the RM 2255 Fully Automated Rotary Microtome (Leica Biosystems, Buffalo Grove, IL, United States). Slides were dried and subjected to Safranin O/Fast green counterstaining (Sigma-Aldrich, St. Louis, MO, United States). Cell nuclei were stained with Hematoxylin solution (Sigma-Aldrich).

The pellets were fixed in a 10% buffered formalin phosphate solution overnight at 4 °C and dehydrated using an increasing ethanol gradient. After soaking in xylene for 2 h and then in liquid paraffin overnight, the samples were embedded in paraffin. The blocks were sectioned at a 6 μm thickness using the RM 2255 Fully Automated Rotary Microtome (Leica Biosystems, Buffalo Grove, IL, USA). The slides were then dried and subjected to Safranin-O/Fast green counterstaining (Sigma-Aldrich). Nuclei were stained with hematoxylin (Sigma-Aldrich). After staining, slides were mounted with a Limonene-Mount mounting medium (Electron Microscopy Sciences, Hatfield, PA, USA) and then covered, and histological staining was performed using a microscope equipped with a color digital camera (Nikon Eclipse E800).