Anti 15 lox

Placenta of patients was collected immediately after caesarean and washed with PBS, with one part was stored in liquid nitrogen, and other parts were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned and stained with haematoxylin and eosin. For immunostaining, slices were boiled in 0.01 mol/L citrate buffer (PH6.0) to retrieve antigen and then naturally cooled to room temperature. Methanol containing 3% H₂O₂ was used to eliminate endogenous peroxidases. Before blocked in 10% goat serum for 30 minutes, the tissue was permeabilized with 0.1% Triton X‐100 for 30 minutes. The sections were incubated with primary antibodies (anti‐FPR2, anti‐5‐LOX, anti‐15‐LOX from Abcam; anti‐PPARγ, anti‐P65 from Santa Cruz) overnight at 4°C, incubated with the secondary antibody at 37°C for 60 minutes and developed with diaminobenzidine tetrahydrochloride. For cell culture slides, secondary antibodies conjugated to fluorescein isothiocyanate (Sigma‐Aldrich) were used.

Placenta tissues were cut into small pieces and homogenized by Potter‐Elvehjem Tissue Grinders on ice, followed by lysed in the RIPA buffer containing 1 mmol/L PMSF, as well as the cells. After quantification by BCA assay, 10 µg protein of each sample was resolved by polyacrylamide gel electrophoresis and transferred to PVDF membranes. Blots were blocked by 5% no‐fat milk for 1 hour and then incubated with specific primary antibodies (anti‐FPR2, anti‐5‐LOX, anti‐15‐LOX from Abcam; anti‐PPARγ, anti‐P65 from Santa Cruz, anti‐IκBα from Servicebio) overnight at 4°C. Blots were incubated with HRP‐conjugated secondary antibody and developed using the ECL system. The protein bands were acquired and quantitated using the Amersham Imager 600(GE) blot documentation instrument provided by the FluoChem M system (Protein Simple).

All procedures were performed as described above [89 (link)]. The antibodies are as follows: anti-FGGY (Abcam), anti-12-LOX (Abcam), anti-15-LOX (Abcam), anti-GPR31 (Abcam), and a biotinylated secondary goat anti-mouse IgG antibody (Santa Cruz), labeled with HRP using a DAB staining kit (Maixin Biotechnology) according to the manufacturer’s instructions. For negative controls, IgG1 was used. Positively stained cells were counted in 5 fields at 200 × magnification.