For immunofluorescence labeling of brain sections, injected mice were perfused with PBS containing 4% paraformaldehyde (PFA). The whole brains were extracted and postfixed in 4% PFA/PBS for 3 days at 4°C and then cryoprotected in 30% (w/v) sucrose in PBS for 3 days, cut horizontally into 40-μm-thick brain sections on a freezing microtome, and stored in PBS/50% glycerol at −20°C. For immunostaining, the floating brain sections were washed briefly in PBS and then permeabilized with 0.2% Triton X-100/TBS for 20 min at room temperature, blocked in 5% normal goat serum (NGS)/PBS for 1 hour at room temperature, and then incubated with primary antibodies diluted in 3% NGS/PBS overnight at 4°C: chicken anti-GFP (1:1000, Aves), mouse anti–human tau Tau13 (1:1000, BioLegend), rabbit anti–human tau TauY9 (1:1000, Enzo Life Sciences), mouse immunoglobulin M (IgM) anti-misfolded tau (1:500, Alz-50, provided by P. Davis), and goat anti-CtB (1:1000, Millipore). After washing three times with PBS, secondary antibodies were diluted in 3% NGS/PBS and applied for 1.5 hours at room temperature: Alexa 488 anti-chicken, Cy3 anti-mouse, Cy3 anti-rabbit, Alexa 647 anti-mouse IgM, and Alexa 647 anti-goat (1:1000, Thermo Fisher Scientific). After three washes in PBS, sections were mounted on microscope glass slides with mounting media containing 4′,6-diamidino-2-phenylindole (DAPI) (Southern Biotech).

Thiazine Red staining of tangles was done by applying 0.05% (w/v) Thiazine Red (Sigma-Aldrich) dissolved in PBS (GIBCO) to permeabilized brain sections for 20 min at room temperature, followed by extensive washing in PBS (four times for 10 min and once overnight) to remove unspecifically bound dye. Imaging of immunolabeled sections was done using 5×, 10×, or 20× objectives on a Zeiss Axiovert equipped with a QuickSnap camera or on an Olympus BX51.

For flow cytometry experiments, directly labeled Tau13–Alexa 647 was produced by incubating anti-human tau antibody (100 μg) Tau13 (BioLegend) with active N-hydroxysuccinimide–Alexa 647 (solved in dimethyl sulfoxide, Thermo Fisher Scientific) for 1 hour at room temperature in PBS (pH 7.5). Excess dye was removed by dialysis of the antibody/dye mix in dialysis cassettes (Slide-A-Lyzer, molecular weight cutoff, 3000; PIERCE) against 2 liters of PBS overnight at 4°C.

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