Rabbit anti fgf21 primary antibody

In this procedure, formalin-fixed, paraffin-embedded mouse livers were incubated with 5% bovine serum albumin (BSA) in 0.05% phosphate-buffered saline (PBS)-Tween for 1 h to permeabilize hepatic cells and block non-specific protein-protein interactions, and was then stained with rabbit-anti FGF21 primary antibody (1:200, Abcam, UK) overnight at 4°C. The secondary antibody (green) was used with DyLight 488 goat anti-rabbit IgG (H+L) buffer (1:250, Abcam, UK) for 1 h. 4′,6-Diamidino-2-phenylindole (DAPI) was used to stain the cell nuclei (blue) (Abcam, UK) before imaging and cell counting [22 (link)].

Hepatic lysate was isolated using radioimmunoprecipitation assay (RIPA) buffer supplemented 1 mM phenylmethylsulfonyl fluoride (PMSF) protein inhibitor (Beyotime Biotechnology, China). Forty micrograms of protein in each lane was electrophoresed with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to 0.2-μm polyvinyldene fluoride (PVDF) membranes (Millipore, MA, USA). After blocking with 5% non-fat milk solution (Yili Industrial Group Co., Ltd, China) for 1 h, the membrane was incubated with diluted rabbit-anti-FGF21 primary antibody (1:1000, Abcam, UK), rabbit-anti-PARP, and cleaved PARP (1:1000, Cell Signaling Technology, USA) overnight at 4°C, developed using a horseradish peroxidase (HRP)-coupled secondary antibody (Beyotime Biotechnology) for 1 h. After adding the chemiluminescent reagent, the membrane was imaged and captured using a gel imager (BIO-RAD, USA). Internal α-tubulin protein (Beyotime Biotechnology) was used as a housekeeping control for target protein normalization [23 (link)–24 (link)].