Anti fabp5

Immunocytochemical staining was conducted on cell-bearing coverslips by the method described elsewhere [15 (link)]. The antibodies used were: Cyclin D1 (Bioss Inc., Beijing, China; 1:100), Tg (Bioss. Inc., Beijing, China; 1:200), E-cadherin (Bioss. Inc., Beijing, China; 1:100), RAR-β (Bioss. Inc., Beijing, China; 1:150), and PPAR-β/δ (Bioss. Inc., Beijing, China; 1:200). Cells incubated in 0.2% DMSO-containing medium were used as a control. The color reaction was performed by using 3,3′-diaminobenzidine tetrahydrochloride (DAB) after the binding of the primary antibody (Vector Laboratories, Burlingame, CA, USA). For double immunofluorescent staining (IF), mouse anti-CRABP2 and rabbit anti-FABP5 were employed in the working concentrations of 1:120. Briefly, the cell-bearing coverslips were washed with phosphate-buffered solution (PBS, pH 7.4), incubated in 3% H₂O₂ for 10 min and then with anti-CRABP2 (1:120; Proteintech, Chicago, IL, USA) and anti-FABP5 (1:120; Proteintech, Chicago, IL, USA) at 4 °C for one night in a humid chamber. Finally, the coverslips were co-incubated with FITC-conjugated goat anti-mouse IgG and PE-conjugated goat anti-rabbit IgG (both 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 37 °C for 60 min in the dark, sealed with fluorescence mounting medium, and observed and imaged under a fluorescence microscope (BX53F, Olympus, Tokyo, Japan).

Immunofluorescence was conducted as previously described [16 (link)]. Antibodies used in this study include anti-RTN3 (Santa Cruz, #sc-374599), anti-FABP5 (Proteintech, #12348-1-AP), anti-DGAT2 (Proteintech, #17100-1-AP), Cy3–conjugated Affinipure Goat Anti-Mouse IgG(H + L) (Proteintech, # SA00009-1), and DyLight 405-labeled Goat Anti-Rabbit IgG(H + L) (Beyotime, # A0605). ImageJ was used to calculate the area of isolated cardiomyocytes. Ten representative images were used per group for analysis.

Western blot analyses were carried out as described previously [17 (link)]. The total protein of the placenta samples was extracted using a Tissue Protein Extraction Kit (CWBIO, Jiangsu, China), and the concentration of the extracted total protein was determined by a bicinchoninic acid assay (CWBIO, Jiangsu, China). Sodium dodecyl sulphate–polyacrylamide gel electrophoresis and transmembrane experiments were carried out with the Mini-PROTEAN Tube Cell instrument (Bio-Rad, Hercules, CA, USA). The membranes were incubated with primary antibodies, including anti-FATP4 (ProteinTech, Rosemont, IL, USA), anti-CD36 (ProteinTech, Rosemont, IL, USA), anti-FABP5 (ProteinTech, Rosemont, IL, USA), and anti-β-actin primary antibodies (Cell Signaling Technology, Danvers, MA, USA). Thereafter, the membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Santa Cruz company, Hercules, CA, USA), and immunoreactive bands were visualized using the ECL kit (Beyotime, Shanghai, China) and documented with a chemiluminescence imaging system (UVP, Upland, CA, USA). The results of the densitometric analysis of bands were quantified by Image J 1.45 software (National Institutes of Health, Bethesda, MD, USA). To calculate the relative expression levels of the target proteins, all blots were standardized to β-actin.