Pepsin solution

Thin coronal sections (6 µm) were taken for histological analysis of caspase-3 activation of chondrocytes in the femoral condyles and tibial plateaus. Sections were heated to 60 °C for 30 min, deparaffinized and hydrated in three changes of xylene and serial alcohol. Antigen retrieval was performed using a pepsin solution (Thermo Fisher Scientific). A rabbit polyclonal antibody against active caspase-3 antibody 9661 (Cell Signaling Technology) or anti-S-nitrosocysteine HY8W12 (Abcam) at 1:000 dilution were added to separate sections and incubated at 4 °C overnight. After three washes with PBS, the sections were incubated with Cy3 goat anti-rabbit IgG (Life Technologies, Molecular Probes^®, (Thermo Fisher Scientific) at 1:000 dilution for 1 h at room temperature, protected from light. The sections were washed five times using PBS, counterstained using Vectashield mounting media with DAPI (Vector Laboratories Inc, Burlingame, CA, USA).

For analysis of the absorption and distribution of LNA ASO in administered mice, a FISH probe targeting to 5’-ASO#26 was designed and purchased from IDT (Supplementary Table 3). For FFPE tissue sections, slides were deparaffinated in Xylene and 100% ethanol and incubated in 1x Universal HIER antigen retrieval reagent (ab208572, Abcam) at 95 °C for 20 min followed by a wash step in 2xSSC buffer. For frozen tissue sections, slides were washed in PBS to remove OCT followed by fixation in fixative (3:1 methanol:acetic acid) for 5 min. Then tissue sections were digested in Pepsin solution (003009, ThermoFisher) at 37 °C for 30 min and dehydrated via 70%, 85% and 100% EtOH. The FISH probe (0.5 μM) was prepared in the hybridization solution (50% formamide, 10% dextran sulfate, 0.1% SDS, 300 ng/ml Salmon Sperm DNA in 2xSSC buffer). The slides incubated with FISH probe were desaturated at 75 °C (for FFPE sections) or 73 °C (for frozen sections) for 5 min and incubated at 37 °C for 16 h in HYBrite (VYSIS). Slides were washed in Wash Buffer 1 (0.3% NP-40 in 0.4× SSC buffer) at 73 °C for exactly 2 min, and then in Wash Buffer 2 (0.1% NP-40 in 2× SSC buffer) at room temperature for 1 min. The nuclei were stained with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher) and slides were observed via LSM710 confocal microscope (Zeiss).