Hrp conjugated donkey anti mouse secondary antibody

Western blot detection of PrP^Sc from brain homogenate was performed as described previously [76 ]. Briefly, brain homogenate (5% w/v) in PMCA conversion buffer is digested with proteinase K (PK) at a final concentration of 100, 200, or 400 μg/ml (Roche Diagnostics Corporation, Indianapolis, IN) at 37°C for 1 or 24 hours. The samples were either enriched for PrP^Sc as described previously [77 (link)] or an equal amount of sample buffer containing 4% (v/v) 2-mercapto ethanol and 8% (w/v) SDS was added and the mixture was incubated at 100°C for 10 minutes. Prion protein was detected with the anti-PrP antibody 3F4 (final concentration of 0.1 μg/ml; Chemicon; Billerica, MA) and HRP-conjugated donkey anti-mouse secondary antibody (Jackson ImmunoResearch; West Grove, PA). The Western blot was developed with Pierce Supersignal West Femto Maximum Sensitivity Substrate according to manufacturer instructions (Pierce, Rockford, IL), imaged on a Kodak 4000R Imaging Station (Kodak, Rochester, NY) and analyzed using Kodak Molecular Imaging Software v.5.0.1.27 (New Haven, CT). Statistical analysis was performed using Prism 6.0 for Mac (GraphPad Software Inc., La Jolla, CA).

Fibroblasts were fixed in 2% paraformaldehyde on ice for 10 minutes. Blocking was performed using 2% BSA in PBS-Tween 0.05%. Mouse monoclonal anti-integrin- α3 (Millipore MAB1952, 1:50) was used and detected with a secondary anti-mouse IgG conjugated with Alexa-488. Images were captured with a Leica epifluorecent microscope and LAS software. For Western blot analysis total protein extracts were separated on 10% Mini-PROTEAN ^® TGX (Bio-Rad, Hercules, CA, USA) and transferred onto a nitrocellulose membrane using Trans-Blot^® TurboTM Mini Nitrocellulose Transfer Pack and Tans-Blot^® Turbo TM Transfer System (BioRad, Richmond, USA). After blocking, membranes were incubated with primary antibodies over night at 4°C. A HRP-conjugated donkey anti -mouse secondary antibody (Jackson Immuno Research) was used. Proteins were detected using chemiluminescence (ECL, Pierce, Rockford, USA). The protein content was expressed in arbitrary units relative to α-Tubulin (Sigma, Missouri, USA) as the protein loading control. The intensity of protein bands was determined by densitometry with ImageJ Software.