4.4. Alcian Blue Staining

Alcian blue staining was used to detect sulfated glycosaminoglycans (sGAGs) in fibrocartilage tissues on serial sections. The sections were deparaffinized in 2 × 10 min changes of xylene and rehydrated in a sequential decrease of ethanol (100%, 96%, 80%, and 70% ethanol). The sections were then rinsed in distilled water and incubated in Alcian blue solution for 15 min. After washing under running tap water for 2 min, samples were rinsed in distilled water and then incubated for 4 min in nuclear fast red solution. The samples were washed under running tap water for 1 min and dehydrated using 95% and 100% ethanol and two changes of xylene. Finally, the sections were mounted using Depex (Serva, serving scientists, Heidelberg, Germany). The expression of sGAG was determined by scoring the intensity in at least three different areas per gliding or traction area of a section per tendon by two independent observers who were blinded to the experiment. The mean was then calculated for all values per section. The completely blue-stained ECM has a maximum score of 10 points, whereas sections without positive staining (only bright-pink stained collagen fibers) received a minimum score of 0.