Immunohistochemical and Immunofluorescence Analysis of SAT Sections

Formalin-fixed, paraffin-embedded SAT sections were used for immunohistochemical analysis and immunofluorescence investigations (n = 5 each), as previously described [23 (link)]. For the immunohistochemical analysis, we used anti-GRP78 (Abcam, Cambridge, MA, USA), Imgenex anti-ATF6 (Novus, Littleton, CO, USA), and anti-EPHX2 antibodies (OriGene Technologies, Inc., Rockville, MD, USA). The immunohistochemical analysis results were quantified using ImageScope software version 11.1 (Aperio, Vista, CA, USA), as previously reported [29 (link)]. For immunofluorescent staining, tissue sections were incubated with anti-GRP78 or anti-ATF6 antibody conjugated with Alexa Fluor-488 (Bioss, Woburn, MA, USA). EPHX2 tissue sections were incubated with anti-EPHX2 antibody (OriGene Technologies, Inc., Rockville, USA) and then incubated with Alexa Fluor-488–conjugated goat anti-mouse secondary antibody (Molecular Probes, ThermoFisher, USA). Nuclei were stained using 0.05% DAPI. The sections were viewed with a Zeiss LSM 710 confocal laser-scanning microscope, and representative areas of the sections were photographed under a 40× objective.