Rabbit serum

The formalin-fixed, paraffin-embedded specimens were cut into 4-μm-thick sections and placed on glue-coated glass slides for immunohistochemistry. Briefly, the samples were deparaffinized in xylene and hydrated in a graded alcohol series and distilled water. Endogenous peroxidase activity was blocked with 3% hydrogen peroxidase for 10 min. Antigen retrieval was performed in citrate buffer (pH 9.0) by autoclaving (Tomy SX-500 High-Pressure Steam Sterilizer, Tomy Seiko Co., Ltd., Tokyo, Japan) and heating at 121 °C for 5 min. The samples were then incubated for 30 min at room temperature (RT) in a blocking solution with 1% rabbit serum (Nichirei Bioscience, Tokyo, Japan). Then, the anti-CD11b (1:100, EPR1244, Abcam, Cambridge, United Kingdom) monoclonal antibody reaction was performed for 16 h at 4 °C. A secondary antibody reaction was performed using an Envision + System-HRP labeled polymer anti-mouse antibody for 30 min at RT, and DAB (3, 3 -diaminobenzidine) was used to visualize the binding of the first antibody. Then, the sections were examined using a Leica DM6000B (Leica, Wetzlar, Germany) fitted with a digital video camera (Leica MC 170HD, Leica). Quantification of CD11b positive cells were counted in ten randomly selected high power (20x) fields in the defect area manually.

Immunohistochemistry (IHC) was performed on 3‐micrometer‐thick sections obtained from paraffin‐embedded samples fixed in 10% formalin. Antigen retrieval for DRP1 was conducted using a pH 6 citrate buffer in a microwave, while p62 antigen retrieval used a pH 9 Tris‐EDTA buffer, and Parkin antigen detection used a pH 8 EDTA buffer. Subsequently, the sections were blocked in 10% rabbit serum (Nichirei Biosciences, Tokyo, Japan). The primary antibodies against DRP1 (Abcam, ab56788, 1:1000), p62 (Abcam, ab207305, 1:2000), and Parkin (Santa Cruz Bio, sc‐32,282, 1:50) were then incubated overnight at 4°C. After blocking endogenous peroxidase activity, the sections were incubated with secondary antibodies (Nichirei Bioscience, Tokyo, Japan). Human kidney, hepatocellular carcinoma (HCC), and gallbladder tissues were used as positive controls for DRP1, p62, and Parkin, respectively.