Casein based protein block

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.

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.