Declere

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.

Five-micron (5 μm) paraffin-embedded sections were deparaffinized and rehydrated. Antigens were retrieved by immersion in DECLERE (Cell Marque, Hot Springs, AR, USA) in moist heat for 15 min. Potential nonspecific binding sites were blocked with 5% normal goat or rabbit serum in PBS. After blocking, the sections are incubated with antibodies specific for MMP-3 (1:25; Santa Cruz Biotechnology, Santa Cruz, CA, USA). After three 5-min washes in PBS, the sections were incubated with biotin-conjugated IgG (Vector Laboratories, Burlingame, CA, USA). A vector-ABC streptavidin-peroxidase kit with benzidine substrate was used for color development. Following counterstaining with diluted hematoxylin, the images were scanned using Aperio Scanscope Console software (Informer Technologies, Inc., Shingle Springs, CA, USA). Sections that were not incubated with the primary antibody served as a negative control. For NIRF imaging, paraffin-embedded sections (without staining) were examined and photographed using a NIRF microscope with 615–675- and 695–790-nm band-pass filters at the Cy5.5 channel.