Hrp conjugated rabbit anti human igg

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).

Western blotting of chimeric MAbs was carried out as described previously^{63 (link)}, except that HRP-conjugated goat anti-mouse IgG (diluted 1:10,000; Sigma, USA) and HRP-conjugated rabbit anti-human IgG (diluted 1:10,000; Abcam, USA) were used for detection. Note that anti-human IgG or anti-mouse IgG secondary antibodies (HRP conjugate) used in this study recognize the constant domains of both heavy and light chains.