Safranin o

After removing the skin, various tendons were macroscopically observed in mice of three genotypes. Hind limbs including Achilles tendon were embedded intact in paraffin after fixation with 4% paraformaldehyde phosphate buffer solution (PBS) for 48 h and decalcification in 18.5% EDT-X (Falma, Tokyo, Japan) for 3 weeks. Tissue sections were sectioned (4.5 μm) in the sagittal or transverse plane. The sections were stained with Hematoxylin and Eosin (MUTO PURE CHEMICALS, Tokyo, Japan), Safranin-O (MUTO PURE CHEMICALS, Tokyo, Japan)/Fast green (Sigma-Aldrich, United States), and Picro-Sirius Red stain kit (ScyTek labolatories inc.). We also measured the transverse-section area of the center of Achilles tendon. Analysis of the transverse section area was carried out using ImageJ software.

Whole-mount Alcian blue (Sigma-Aldrich, United States) and Alizarin red S (Sigma-Aldrich, United States) staining of skeletons were performed on Control, Cart-miR23clus KO and Glob-miR23clus KO mice at postnatal day 0. All knee joints were embedded intact in paraffin after fixation in 4% paraformaldehyde phosphate buffer solution (PBS) and decalcification in K-CX or EDT-X (FALMA, Japan). Knee joints were sectioned (4.5 μm) in the coronal plane anterior to posterior through the central weight-bearing region of the femorotibial joint. The sections were stained with Safranin O (MUTO PURE CHEMICALS, Tokyo, Japan) and Fast Green (Sigma-Aldrich, United States) and three different sections per joint were analyzed microscopically. Three different researchers were blinded while performing all manual scorings. Damage to the articular cartilage (maximum of 24 points per knee joint section; 6 points for each quadrant of the tibial/femoral cartilage) was evaluated using the OARSI scoring system (Glasson et al., 2010 (link)). Subchondral bone changes, meniscus degradation and the severity of synovitis were evaluated using the right knee joints according to previously described histopathological scoring systems (Krenn et al., 2002 (link); Kwok et al., 2016 (link); Nagira et al., 2020 (link)).