α fasn

Immunohistochemistry on mouse liver sections was carried out as described previously (11 (link)) using heat denaturation in citrate buffer (pH 6.0) to expose the epitope. Stained slides were digitized using an Allied Vision Technologies Stingray F146C color slide scanner and quantified using the Spectrum Plus Image analysis software (Aperio). Antibodies were used in immunohistochemistry as follows: Ki67 (1:100; LabVision, catalog no. RM9106), α-BNIP3 (1:100; Sigma Prestige, HPA003015), α-FASN (1:100; Cell Signaling Technology, catalog no. 3180), and α-ACACA (1:200; Cell Signaling Technology, catalog no. 3676). Oil Red O staining was performed on frozen liver sections that were warmed to RT, fixed for 10 min in cold 10% neutral buffered formalin, and allowed to air dry. Slides were incubated in propylene glycol for 3 min and then in Oil Red O/propylene glycol solution for 10 min, followed by 3 min in 85% propylene glycol and washing three times in water. Oil Red O reagents were obtained from Newcomer Supply (catalog no. 9119A). Oil Red O–stained sections were then counterstained in hematoxylin and mounted using Vectashield (Vector Laboratories). Oil Red O droplets were quantified in the red channel following deconvolution and thresholding, using ImageJ (NIH).