Immunohistochemical Analysis of AMPK Signaling

Immunohistochemistry was performed using a Discovery Auto-Stainer with automated protocols (Ventana Medical Systems, Inc., Tucson, AZ, USA; Roche, Mannheim, Germany; Ventana Discovery XT, NexES version 10.6 software, DAB-Map kit 760 − 124), as previously described [22 (link), 28 (link)]. The pAMPK antibody (rabbit monoclonal, 40H9, 2535, and 1:300) was purchased from Cell Signaling Technology, Inc. (MA, USA). Total AMPK antibody (mouse monoclonal, D-6, sc-74,461, 1:50), PPARg antibody (mouse monoclonal, E-8, sc-7273, 1:50), and PPARa antibody (mouse monoclonal, H-2, sc-398,394, 1:50) were purchased from Santa Cruz Biotechnology Inc (TX, USA). We have previously clarified the specific immunostaining for pAMPK and total AMPK antibodies in the liver and heart [24 (link)]. We performed negative control staining and confirmed that no primary antibodies were detected in tissues without secondary antibodies.