Mouse anti aβ 4g8 antibody

Samples of sortase reactions with Aβ were added to 4x LDS buffer and, without heat denaturing, loaded onto a 4–12% bis-tris gel and ran at 160 V for 30 min in MES running buffer. Gels were transferred to PVDF membrane via iBlot and membrane blocked with Superblock Blocking Buffer (Thermofisher, 37515) for 1 h at rt. The membrane was then incubated with mouse anti-Aβ 4G8 antibody (Biolegend, 800702, 1:1000 dilution in Superblock TBS + 0.1% tween-20) overnight at 4°C followed by washing in PBS-T (PBS + 0.1% tween-20) three times for 5 minutes each. Secondary antibodies Streptavidin-IR800 (Licor, 926–32230) and goat anti-mouse-IR680LT (Licor, 926–68020) (both 1:10,000 dilution in Odyssey Block in PBS (Licor, 927–40000), 0.1% Tween-20, 0.01% SDS) were applied for 30 min at room temperature in the dark. The membrane was washed with PBST three times for 5 minutes each, followed by one wash with MilliQ water and imaged on an Odyssey Imager.

Brains were immersion-fixed in 10% formalin, then dehydrated and embedded in paraffin. Serial 10 µm-thick sagittal sections were cut using a microtome. For Aβ staining, brain slices were previously treated with 85% formic acid for 5 min at room temperature for antigen retrieval followed by a blocking step with 3% H₂O₂/10% methanol solution in PBS. Then, brain sections were incubated overnight with mouse anti-Aβ 4G8 antibody (Biolegend, Cat#800710) diluted 1:1,000 in PBS/0.02% Triton-X 100 (Sigma, St. Louis, MO, USA) and then incubated for 1 h with sheep anti-mouse IgG–Horseradish Peroxidase (HRP) secondary antibody (GE Healthcare, Little Chalfont, UK) diluted at a 1:500 ratio. Peroxidase reaction was visualized using 3,3′-Diaminobenzidine (DAB) Kit (Vector Laboratories, Burlingame, CA, USA) following manufacturer’s instructions. Stained sections were dehydrated in graded ethanol, cleared in xylene, and cover-slipped with DPX mounting medium (Innogenex, San Ramon, CA, USA). Brain tissue slices from an AD patient were counterstained with hematoxylin. ThS staining was performed in brain slices consecutive to 4G8 stained sections. For this, tissue slices were incubated with a ThS (Sigma, St. Louis, MO, USA) solution (0.1% ThS in 50% ethanol) for 15 min after deparaffinization. After incubation, sections were washed for 2 min in 80% ethanol, dehydrated and cover-slipped.