Ab181114

Placental tissues embedded in wax were sliced at a thickness of 5 μm. For the hematoxylin and eosin (H&E) assay, the slides were stained with hematoxylin for 5 min and eosin Y for 30 s (C0105S, Beyotime, Beijing, China). Images were captured using an Olympus DP71 microscope. For immunohistochemistry assay, antigen retrieval was carried out in citrate buffer pH 6.0 (P0083, Beyotime, Shanghai, China) using a pressure cooker for 3 min, followed by blocking endogenous peroxidases using 0.3% H₂O₂ for 15 min. After blocking 20 min by blocking buffer (P0260, Beyotime, Shanghai, China), the slides were incubated overnight at 4°C with an anti-CPT2 antibody (1:150, ab181114, Abcam, Cambridge, UK), anti-CPT1b antibody (1:150, 22170-1-AP, Proteintech, Wuhan, China). On the next day, the slides were washed and then incubated with a secondary antibody (PV9001, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) for 20 min at room temperature. The slides were incubated with DAB (ZLI9018, Zhongshan Gold Bridge Biotechnology Co, Beijing, China) to detect side-specific antigen-antibody binding, followed by staining with hematoxylin. Then, the slides were dehydrated and sealed with neutral gum. Images were captured using an Olympus DP71 microscope. Five random views per tissue section were used to quantify the mean IOD (IOD/area) using Image-Pro Plus 6.0.

Kidney slices were fixed in alcohol–formalin–acetic acid, dehydrated with ethanol and xylene, embedded in paraffin, and cut into 3 μm sections. Samples were then deparaffinized, rehydrated, and heated for 20 minutes at 97°C in citrate buffer. Endogenous peroxidase was inactivated by incubation for 10 minutes at room temperature in 0.3% H₂O₂. Sections were incubated with PBS containing 1:50 anti-Hspa5 (catalog sc-1050, Santa Cruz Biotechnology) and anti-Cpt2 (catalog ab181114, Abcam). Next, sections were incubated with anti-rabbit or anti-goat antibodies conjugated with peroxidase-labeled polymer [Dako, Agilent Technologies, EnVision+ Dual Link System-HRP (DAB+) catalog K4065], then visualized with a peroxidase kit (Dako). Finally, the tissue sections were counterstained with hematoxylin.