Serum free protein blocking reagent

Fresh frozen AD brain sections with no fixation were exposed to antigen retrieval citrate buffer (Target Retrieval Solution, Dako, Santa Clara CA, USA) for 20 min and incubated in a humidified chamber with serum-free protein blocking reagent (Dako) for 1 h to block non-specific staining. The sections were incubated overnight at 4 °C with primary antibodies (muPMN310, huPMN310, aducanumab, bapineuzumab, isotype controls) at 1 µg/ml and washed 3 times for 5 min in TBS containing 0.1% Triton-X-100 (TBS-T) buffer. Secondary HRP-conjugated rabbit anti-human IgG (0.4 μg/ml; Abcam, San Francisco CA, USA) or sheep anti-mouse IgG (1 μg/ml; GE Healthcare, Chicago IL, USA) antibodies were added to the sections and incubated for 1 h, followed by 3 washes in TBS-T buffer. Secondary antibody was also added to sections that were not exposed to primary antibody as a negative control. The HRP enzyme substrate, biaminobezidine (DAB) chromogen reagent (Vector Laboratories, Burlingame CA, USA), was then added to the sections followed by rinsing with dH₂O. The sections were counterstained with haematoxylin QS (Vector Laboratories, Burlingame CA, USA). The slides were examined under a light microscope (Zeiss Axiovert 200 M, Carl Zeiss Toronto ON, Canada) and representative images were captured using a Leica DC300 digital camera and software (Leica Microsystems Canada Inc., Vaughan ON, Canada).

Fresh frozen brain sections with no fixation were exposed to antigen retrieval citrate buffer (Target Retrieval Solution, Dako, Santa Clara, CA, USA) for 20 min and incubated in a humidified chamber with serum-free protein blocking reagent (Dako) for 1 h to block non-specific staining. The sections were incubated with test mAbs, a pan α-Syn-reactive antibody (clone 4D6, BioLegend), or mIgG1 isotype control at 20 µg/mL overnight at 4 °C. Sections were then washed three times in TBS with 0.1% Triton-X-100 (TBS-T) followed by incubation with HRP-conjugated sheep anti-mouse IgG secondary antibody (Cytiva, Vancouver, BC, Canada, NA931) for 1 h at room temperature and three washes in TBS-T. Secondary antibody was also added to sections that were not exposed to primary antibody as a negative control. The HRP enzyme substrate, biaminobezidine (DAB) chromogen reagent was then added to the sections, followed by rinsing with distilled water. The sections were counterstained with haematoxylin QS (Vector Laboratories, Burlingame, CA, USA). The slides were examined under a light microscope (Zeiss Axiovert 200 M, Carl Zeiss Toronto, ON, Canada) and representative images were captured using a Leica DC300 digital camera and software (Leica Microsystems Canada Inc., Vaughan, ON, Canada).

Each excised xenograft tumor was fixed by formaldehyde, embedded in paraffin, and then sliced on glass slides. For immunohistochemistry, paraffin-embedded tumor section was deparaffinized, rehydrated, stained with hematoxylin (Dako) and eosin (Sakura Finetek), and analyzed by IHC using antibodies as described above. Briefly, the tumor sections were treated with xylene and ethanol, and incubated with Antigen Retrieval Solution pH9 (Nichirei) to retrieve antigens. Then, tumor sections were incubated with 3% H₂O₂ solution (Wako) for peroxidase blocking and Serum-free Protein Blocking Reagent (Dako), according to manufacturer's instructions. These sections were incubated with primary antibodies diluted in the Antibody Diluent Solution (Dako), followed by incubation with Histofine Simple Stain MAX PO (Nichirei), and then stained with substrate-chromogen (Histofine DAB kit, Nichirei). Finally, tumor sections were counterstained with hematoxylin (Dako) and observed in a bright-field microscope (Leica).