Safranin o solution

Mice feet were fixed in 4% paraformaldehyde and embedded in paraffin. Hematoxylin and eosin (H&E) staining with tissue sections was performed using standard techniques [33 (link)]. For safranin O staining, deparaffinized slides were rehydrated and incubated with Hematoxylin QS solution (Vector Laboratories, Burlingame, CA, USA) for 5 min. After washing in running tap water for 5 min, slides were destained quickly in acid ethanol and washed again. Staining with 0.001% Fast green FCF solution (Sigma-Aldrich, St Louis, MO, USA) was conducted for 5 min, and slides were rinsed with 1% acetic acid solution for 10 s. After staining with 0.2% safranin O solution (Merck Millipore, Burlington, MA, USA) for 5 min, slides were dehydrated and mounted. All experimental processes were performed at room temperature.

Deparaffinized sections (3–5 μm) were Haematoxylin Eosin (HE) stained using a standard protocol. For Alcian Blue (AB) staining, the sections were incubated for 3 minutes in 1 % acetic acid and then stained 15 minutes in 1 % AB (Carl Roth GmbH). Subsequently, they were rinsed in 3 % acetic acid and cell nuclei counterstained using nuclear fast red aluminium sulfate solution (Carl Roth GmbH) for 5 minutes. For the safranin-O staining the slides were incubated for 10 minutes in a Weigert’s iron hematoxylin (Carl Roth GmbH) and afterwards rinsed in running tap water for 10 minutes. Subsequently, the sections were stained in 0.001 % fast green solution (Sigma-Aldrich) for 5 minutes. After rinsing them briefly in 1 % acetic acid the slides were stained in 0.1 % safranin-O solution (Merck KGaA) for up to 5 minutes. Dehydrated sections were mounted with Entellan mounting medium (Merck KGaA). The stained sections were further analyzed by applying score systems by two observers. To consider species-dependent peculiarities in rats two scoring systems for OA were used, the well-established Mankin Score, which was originally designed for human cartilage and the score system adapted by Glasson et al., [10 (link)] suitable for the very thin rodent joint cartilage. The scoring system according to Krenn et al., [11 (link)] was applied to classify synovitis.

Spheroids were fixed in 4% paraformaldehyde (PFA) for 24 h at room temperature, cryo-protected in 30% sucrose (4°C, overnight), included in optimal cutting temperature (OCT) compound, and cut in 10 μm-thick slices through a cryostat microtome (mod. CM 1950, Leica, Wetzlar, Germany). Safranin-O staining was used to detect acidic proteoglycans present in the ECM, according to standard methods (Schmitz et al., 2010b (link)). In more detail, slices were stained for 10 min with Wiegert’s Iron Hematoxylin (Biognost), 5 min with 0.5 g/L Fast green (Sigma-Aldrich), and 7 min with 0.1% Safranin-O solution (Merck Millipore, United States). Samples were dehydrated with an increasing ethanol gradient (70%–75%–95%–100%) and cleared in xylene for 2 min. Sections were then mounted using Eukitt (Sigma-Aldrich) mounting medium. Safranin-O brightfield images were acquired with a microscope (BX53, Olympus, Tokyo, Japan) equipped with an Olympus SC180 camera (Tokyo, Japan) and Olympus U-TV0.5XC-3 camera adapter operating Olympus cellSens standard 3.2 software.