Safranin o fast green staining

After KOA induction, the rabbits were anesthetized by intraperitoneal injection with 3% sodium pentobarbital solution (1.5 mL/kg). For X-ray examination, the anteroposterior radiographs were taken in the supine position with hip flexion at 30°, knee extension at 0°, hip abduction at 15°, and keeping the patella in right forward. The radiating tube was 110 mm away from the knee joint. The left hind leg was extended and placed in the decubitus position, while the right hind leg was bent at 70°, and the knee was bent at 45°. The parameters for X-ray examination were set as follows: tube voltage 50 kV, current 250 mA, exposure strength 32 mAs, and exposure time for 128 ms.
For MRI (MAGNETOM Prisma 3.0T, Siemens, Germeny) examination, the rabbits were kept in the supine position and the knee was adjusted to a valgus position with a fixed angle of 15°. The patella was kept at the center of the scanner. The parameters for MRI examination were set as follows: (1) T1-tse-cor (FOV:100 × 100 mm、ST:2 mm、TR:831 ms、TE:11 ms); (2) T2-tse-sag (FOV:100 × 100 mm、ST:2 mm、TR:6860 ms、TE:84 ms).
The histomorphological observation of femoral condyle cartilage in rabbits was performed with hematoxylin-eosin (HE) staining and safranin O-fast green staining using standard protocols (Solarbio, Beijing, China).

At 6 weeks after treatment, the rats were sacrificed, and articular cartilage samples were collected. After fixation with paraformaldehyde and decalcified in 10% EDTA, the tissues were embedded in paraffin and sectioned into a 5 μm thick section. Sections were made perpendicular to the defect area. The selected sections were deparaffinized in xylene, rehydrated through a graded series of ethanol washes, and followed by hematoxylin and eosin (H&E) and Safranin O/Fast Green staining (Solarbio). Then, the sections were stained with freshly prepared Weigert solution for 5 min and differentiated with acid differentiation solution for 15 s. The sections were immersed in Fast Green and Safranin O staining solution for 5 min and 2 min, respectively, and washed with distilled water for 1 min. The sections were washed with weak acid solution for 1 min and washed with distilled water for 1 min. The sections were dehydrated and transparent and finally observed under a light microscope.

Part of the excised synovial samples of every patient were immediately fixed in 10% formalin for 16–24 h. The samples were then embedded in paraffin wax and sectioned into 4–6 µm for further Hematoxylin and eosin (H&E) staining, Masson’s trichrome staining, and Safranin O-Fast Green staining (Solarbio, China) according to the manufacturer’s protocol.
Immunohistochemistry (IHC): After deparaffinization and dehydration, the sections underwent antigen retrieval in citrate buffer, quenched in 3% H₂O₂, and blocked with goat serum. The sections were further incubated with the specific antibody against COL 1 (Abcam, United Kingdom) and COL 3 (Abcam, United Kingdom) overnight at 4°C. SP Rabbit and Mouse HRP Kit (DAB) (CW Biotech, China) were used to detect specific labeling according to the manufacturer’s protocol. Hematoxylin (Solarbio, China) was used for counterstain sections.