Tissue processing and Hematoxylin and Eosin (H&E) staining were performed at Michigan State University Investigative
HistoPathology Laboratory. In short, formalin-fixed specimens were sectioned (4μ), deparaffinized, and underwent
heat-induced epitope retrieval in Citrate Plus buffer (ScyTek Laboratories, UT, USA). Endogenous peroxidase activity was blocked
with 3% hydrogen peroxide/methanol bath for 30 minutes. Standard micro-polymer complex staining steps were performed at room
temperature on the intelliPATH FLX® automated stainer, and all staining steps were followed by rinses in TBS automation
wash buffer (Biocare Medical, CA, USA). Sections were blocked with non-specific protein with Background Punisher (Biocare Medical,
CA, USA) for 10 minutes, and then incubated with specific Rabbit anti – Ucp1 (Alpha Diagnostics, TX, USA) in normal
antibody diluent (Tris Buffered) for 30 minutes (ScyTek Laboratories, UT, USA). Micro-polymer reagents were subsequently applied
for specified incubations followed by reaction development with Romulin AEC™ chromogen and counterstained with CAT
Hematoxylin (Biocare Medical, CA, USA).
HistoPathology Laboratory. In short, formalin-fixed specimens were sectioned (4μ), deparaffinized, and underwent
heat-induced epitope retrieval in Citrate Plus buffer (ScyTek Laboratories, UT, USA). Endogenous peroxidase activity was blocked
with 3% hydrogen peroxide/methanol bath for 30 minutes. Standard micro-polymer complex staining steps were performed at room
temperature on the intelliPATH FLX® automated stainer, and all staining steps were followed by rinses in TBS automation
wash buffer (Biocare Medical, CA, USA). Sections were blocked with non-specific protein with Background Punisher (Biocare Medical,
CA, USA) for 10 minutes, and then incubated with specific Rabbit anti – Ucp1 (Alpha Diagnostics, TX, USA) in normal
antibody diluent (Tris Buffered) for 30 minutes (ScyTek Laboratories, UT, USA). Micro-polymer reagents were subsequently applied
for specified incubations followed by reaction development with Romulin AEC™ chromogen and counterstained with CAT
Hematoxylin (Biocare Medical, CA, USA).