Mouse igg isotype control

Following deparafinization and rehydration endogenous peroxidases were blocked by immersion in 0.3% v/v hydrogen peroxide in methanol for 20 minutes. The slides were rinsed thoroughly with tris buffered saline containing 0.05% v/v Tween 20 (TTBS) and incubated for 30 minutes with 10% v/v normal horse serum in TTBS at room temperature. Slides were incubated at 4°C overnight with anti prion antibody 3F4 (2.6 μg/mL: Millipore, Temecula, CA) with 3% v/v normal horse serum in TTBS. Slides were rinsed with TTBS and incubated with biotinylated horse anti-mouse secondary antibody (1 μg/mL: Vector laboratories, Burlingame, CA) in 3% v/v normal horse serum TTBS for 30 minutes at room temperature. Signal amplification was performed using the Vectastain Elite ABC-HRP (Vector, Burlingame, CA) and diaminobenzidine reaction was used to visualize antigen location. The following controls were used to ensure specificity of IHC: use of a mouse IgG isotype control (Abcam, Cambridge, MA) in place of primary antibody and omission of either the primary or secondary antibodies with all other steps being the same. Lymphoid tissue sections not further than 140 μm apart were processed for PrP^C and examined using a Nikon Eclipse 80i light microscope. Images were captured with an Infinity 2 digital camera (Lumenera, Ottawa, ON) and ImageJ software (NIH, Bethesda, MD).