Paa626ra01

Knee joint specimens of Guinea pig were fixed and decalcified and then embedded in paraffin. The slices were then cut into 6-μm sections and mounted on slides, and the degeneration degree of tibial plateau cartilage tissue of knee joint was observed after safranin O staining and compared between groups with Osteoarthritis Research Society International (OARSI) score [12 ].IHC staining was used for detection of Matrix Metalloproteinase-13 (MMP-13, 1:100,
PAA099Ra01,Cloud-Clone Corp.,USA) and Caspase-3 (1:200,PAA626Ra01,Cloud-Clone Corp.,USA). We quantitatively scored the IHC results accordingto the percentage of positive chondrocytes and the staining intensity, as described below. We rated the intensity of staining on a scale of 0 to 3: 0, negative; 1, weak; 2, moderate; and 3, strong. We assigned the following proportion scores: 0 if 0% of the chondrocytes showed positive staining, 1 if 0 to 1% of the chondrocytes were stained, 2 if 2 to 10% were stained, 3 if 11 to 30% were stained, 4 if 31 to 70% were stained, and 5 if 71 to 100% were stained. We then combined the proportion and intensity scores to obtain a total score (range: 0–8), as described previously [13 (link)]. The results were assessed by 2 experienced pathologists a blinded manner.

Rabbit polyclonal primary antibodies for caspase-3 (PAA626Ra01), NF-κB (PAB824Ra01), TNF-α (PAA133Ra01), IL-1β (PAA563Ra01) and IL-10 (PAA056Ra01) were purchased from Cloud-Clone Corp, Katy, TX, USA. Dako DAB+ substrate chromogen (K3468) and Dako EnVision™+ System/HRP (K4003) were purchased from Agilent Technologies, Inc., USA. Metformin was purchased from Hovid Bhd, Malaysia, while STZ was purchased from Sigma-Aldrich (USA). All other reagents were of analytical grade.

Testicular sections of 5 µm were used for caspase-3 and PCNA immunostaining. Tris–EDTA buffer with 0.05% tween 20, pH 9.0 was used for antigen retrieval in a pressure cooker for 5 min. Thereafter, endogenous peroxidase blocking was performed using 3% H₂O₂ in PBS for 5 min. Sections were incubated overnight at 4 °C with rabbit polyclonal primary antibodies for caspase-3 (PAA626Ra01, 1:150) and PCNA (PAA591Mi01, 1:50) (Cloud-Clone Corp, Houston, TX, USA) after rinsing with distilled water and tris-buffered saline containing 0.05% tween 20 (TBST, pH 8.4). Sections were rinsed twice for 5 min with TBST and incubated at room temperature for 30 min using Dako EnVision + System/HRP-labelled polymer containing goat anti-rabbit secondary antibody (catalogue number: K4003, Agilent Technologies, Inc. Santa Clara, USA). DAB substrate (Agilent Technologies, Inc. Santa Clara, USA) was then utilised for detection. Sections were counter-stained with haematoxylin and viewed under a light microscope (Olympus BX41, Olympus Corporation, Tokyo, Japan). ImageJ software (NIH-Bethesda, MD, USA) was used to analyse the intensity of staining and percentage positive cells, for both caspase-3 and PCNA proteins. The results were expressed as fold change relative to the normal control group.