Hrp conjugated sheep anti mouse igg secondary antibody

CpG 1018 was custom-synthesized by Trilink Biotechnologies (San Diego, CA, USA). AddaVax was obtained from InvivoGen (vac–adx–10, San Diego, CA, USA). Reagents used in molecular cloning, such as Phusion DNA polymerase and restriction enzymes, were purchased from New England Biolabs (NEB, Ipswich, MA, USA). Fluorescence-conjugated antibodies used in immunostaining and flow cytometry were obtained from BioLegend (San Diego, CA, USA). TMB substrate was purchased from Thermo Fisher Scientific (34028, Waltham, MA, USA). Horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG secondary antibody was purchased from Cytiva (NA931, Marlborough, MA, USA). HRP-conjugated anti-mouse IgG1 was purchased from Invitrogen (046120, Waltham, MA, USA). HRP-conjugated anti-mouse IgG2c was purchased from Bethyl Laboratories (A90–136P, Montgomery, TX, USA).

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).