Arrdc3 antibody

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.

Cells were lysed in cold RIPA-EDTA buffer [50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40; 0.5% sodium deoxycholate; 0.1% SDS; and 5 mM EDTA] containing 1 mM phenylmethylsulfonyl fluoride, 1 mM Na₃VO₄, and protease inhibitor (Thermo Scientific, Rockford, IL). The protein concentrations were determined using the BCA protein assay kit (Thermo Scientific). The samples were separated on 4% to 20% gradient SDS PAGE. The separated proteins were transferred to PVDF transfer packs using the Trans-Blot Turbo transfer system (Bio-Rad, Hercules, CA). The blots were incubated with primary antibodies in TBS-T or TBS-T with 5% w/v nonfat dry milk overnight at 4°C, and then with appropriate secondary antibodies conjugated to IgG-horseradish peroxidase. CRM-1 (H-300) and beta-actin (C-11) antibodies were purchased from Santa Cruz Biotechnology. ARRDC3 antibody was obtained from Abcam. Proteins were detected using a Clarity Western ECL substrate (Bio-Rad). All bands were imaged with ChemiDoc Touch Imaging System (Bio-Rad).