Hematoxylin 1

Formalin fixed paraffin embedded (FFPE) tissue blocks were sectioned at 4 microns, and deparaffinized through three changes of xylenes and a decreasing series of ethanol. Antigen retrieval was performed in a steam cooker for 15 minutes in Declere (Cell Marque) working solution. Endogenous hydrogenase was blocked by incubation in 3% Hydrogen Peroxide for five minutes. Slides were incubated in Casein-based protein block (Biogenex) for 20 minutes before incubation with ARRDC3 antibody (Abcam) at room temperature for 30 minutes. Slides were then rinsed with buffer and incubated with Amplifier from Hi-Def Polymer Detection Kit (Cell Marque) for 10 minutes at room temperature. Afterwards slides were rinsed with buffer and incubated in DAB chromogen for six minutes at room temperature for color development. The slides were counterstained with Hematoxylin I (Richard Allan Scientific), rinsed in water, and dehydrated through a series of increasing ethanol and three changes of xylenes. Slides were then coverslipped. Digital images of slides were generated via Aperio AT scanner at 20×. Immunohistochemistry assays were performed on a Biogenex I6000 automated stainer. The apoptag apoptosis assay (Millipore, Cat No. 7100) and Masson's Trichrome (Polyscientific) staining were performed manually per the manufacturer's instructions.

Stearic acid (≥98.5%), oleic acid (≥99%), linoleic acid (≥99%), diatomaceous earth, insulin, dexamethasone, 3-isobutyl-1-methyl-xanthine, and fatty acid free bovine serum albumin (BSA) were obtained from the Sigma-Aldrich Chemical Co. (St. Louis, MO). Trypan blue was purchased from Eastman Kodak Company (Rochester, NY). Oil Red O was acquired from Rowley Biochemical (Rowley, MA) and Hematoxylin I was obtained from Richard-Allan Scientific (Kalamazoo, MI).

Formalin-fixed paraffin-embedded (FFPE) tissue blocks were sectioned at 4 μm, and deparaffinized through three washes in xylene and a decreasing series of ethanol. Antigen retrieval was performed in a steam cooker for 15 minutes in Declere (Cell Marque) working solution. Endogenous hydrogenase was blocked with 3% hydrogen peroxide for five minutes. Slides were incubated in casein-based protein block (Biogenex) for 20 minutes before incubation with the respective antibodies at room temperature for 30 minutes. Slides were then rinsed with buffer and incubated with Amplifier from Hi-Def Polymer Detection Kit (Cell Marque) for 10 minutes at room temperature. Afterwards slides were rinsed with buffer and incubated in DAB chromogen for six minutes at room temperature for color development. The slides were counterstained with Hematoxylin I (Richard Allan Scientific), rinsed in water, and dehydrated through a series of increasing ethanol and three changes of xylene. Slides were mounted on coverslips. Digital images of slides were generated via Aperio AT scanner at 20×. Immunohistochemistry assays were performed on a Biogenex i6000 automated stainer. Masson's Trichrome (Polyscientific) staining was performed manually as per the manufacturer's instructions.

After baking at 60°C, the tissue cleared and rehydrated, 5. Before staining with Hematoxylin 1 (Thermo, Cat#7221), followed by Clarifier, Bluing Reagent (Thermo, Cat# 7301) and finally Eosin-Y (Thermo, Cat# 7111).