Biotinylated horse anti mouse secondary antibody

Mast cells were stained with an anti-mast cell protease 2 (MCPT2) antibody (1:500; Moredun Scientific, Penicuik, Scotland, UK). After deparaffinization and rehydration, endogenous peroxidase activity was inhibited with 5% H202 for 30 min and nonspecific binding sites were blocked with a protein-blocking solution (Dako, Glostrup, Denmark). Slides were incubated overnight with MCPT2 antibody at 4 °C, then 1 h at room temperature with a horse anti-mouse biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, CA, USA). Finally, slides were incubated with 3,3′-diaminobenzidine (DAB substrate development kit, Vector Laboratories) for 3 min. The number of MCPT2-positive cells was counted in 5 nonoverlapping high-power fields per slide, 1 slide per animal, and expressed as the number of MCPT2-positive cells per area of lamina propria.

Primary cortical neuron-BV2 co-cultures were fixed by incubating the cells in 4% PFA for 20 min at RT. Subsequently, cells were permeabilized in methanol containing 0.3% H₂O₂ for 10 min. Prior to MAP2 staining, nonspecific binding was inhibited by incubation with PBS containing 1% BSA and 10% horse serum (Vector Labs, Orton Southgate, Peterborough, UK) for 20 min, at RT. Neuronal cells were immunolabeled with anti-MAP2 antibody (1:2000, Sigma-Aldrich, St. Louis, MO, USA, M9942) overnight at 4 °C. Next, cells were incubated with biotinylated horse anti-mouse secondary antibody (1:200, Vector Labs, Orton Southgate, Peterborough, UK) for 1 h. ExtrAvidin-HRP tertiary antibody staining (1:500, Sigma-Aldrich, St. Louis, MO, USA) was performed for 1 h, at RT. To develop the staining, cells were incubated with the ABTS peroxidase substrate (Vector Labs, Orton Southgate, Peterborough, UK) for 30 min. From each well, 150 μL of substrate solution was collected and absorbances was measured at 405 nm with a microplate reader (BioRad, Hercules, CA, USA). Levels of secreted TNF-α were determined from 1:2 diluted conditioned culture media with a Ready-SET-Go mouse TNF-α kit (eBioscience, San Diego, CA, USA).

From each formalin-fixed and paraffin-embedded sample, 5 μm-thick sections were cut. Histological sections were deparaffinized and rehydrated using graded alcohols, and endogenous peroxidase activity was blocked for 30 min in a 0.3% H₂O₂ methanol solution. Heat-induced antigen retrieval was performed using a pressure cooker for 20 min in a citrate buffer (pH 6.0). After washing in Tris-buffer, protein blocking was performed using normal horse serum for 30 min at 25°C. Sections were then incubated with a mouse anti-human Ki-67 primary antibody (MIB-1; Dako, CA, USA) diluted 1:600 in Tris buffer for 18 h at 4°C. After rinsing sections in Tris buffer, slides were incubated with a biotinylated horse anti-mouse secondary antibody (Vector Laboratories, CA, USA) for 30 min at 25°C. Immunohistochemical signals were detected using an avidin-biotin system (Vector Laboratories, CA, USA) and 3-amino-9-ethylcarbazole (AEC) substrate-chromogen kit (Vector Laboratories, CA, USA). Sections were counterstained with Harris hematoxylin and mounted using an aqueous mounting medium (Aquatex, Sigma-Aldrich, MO, USA). The Ki-67 value was expressed as the percentage of positively stained cells, calculated by counting 1,000 cells per section (400× magnification).

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).

To analyze the effect of PA96 in dopamine neurons in vivo, tyrosine hydroxylase immunohistochemistry was performed as described previously [38 (link)]. The PFA-fixed and paraffin-embedded brains were cut into 5 µm thick sections (3 sections per slide), and every fifth slide was taken for TH staining. The sections were deparaffinized, followed by a citrate antigen retrieval procedure. Endogenous peroxidase was inactivated by 30 min incubation with 3% H₂O₂. The sections were blocked with 5% normal goat serum and then probed with monoclonal TH antibody (1:2000, Millipore Cat# MAB318 Lot# RRID:AB_2201528) overnight at +4 °C. The sections were washed, and biotinylated horse antimouse secondary antibody (1:200, Cat# BA-2001, Vector Laboratories, Burlingame, CA, USA) was applied for 1 h, followed by washing. The sections were treated with ABC and DAB staining kits (Vector Laboratories, CA, USA) according to the manufacturer’s instructions to visualize bound antibodies.