Gill s haematoxylin solution

Deparaffinized and rehydrated sections were first stained in Gill's haematoxylin solution (Sigma, stl Louis) for 10 minutes and then rinsed in running tap water for 5 minutes and incubated in alkaline sodium chloride solution for 20 minutes. Sections were then stained in Congo red working solution for 15 minutes (0.2 % in 80 % ethanol saturated with sodium chloride; Sigma), followed by dehydration through 95 % alcohol. They were then dehydrated, hyalinized and mounted for microscopic examination.

Mice were sacrificed as described above and the TA was dissected from the ventral crest of the tibia and mounted vertically with the largest part of the TA on the base of a cork disc (Fischer). The proximal tendons of the TA were used to balance the TA upright against a needle to enable transverse cross-sectioning. The TA was coated in Tissue Tek (Leica) and snap frozen in liquid nitrogen-cooled iso-pentane (VWR). Transverse cross-sections (10 μm) were generated on a Leica Cryostat (Leica) and collected on pre-treated positively-charged glass microscope slides (VWR). Transverse cross-sections of the TA were stained with filtered Gill’s haematoxylin solution (Sigma-Aldrich) and eosin (VWR) to analyse tissue morphology, or Weigert’s haematoxylin (TCS Biosciences) and Van Gieson solution (Sigma-Aldrich) to analyse fibrosis, according to standard protocols. Sections were dehydrated in ethanol, immersed in xylene (VWR), mounted with a glass coverslip in Entellen (Merck) and air-dried under a laminar flow cupboard.