Anti aβ

To obtain autoradiographic information at the cellular resolution level, frozen cryostat sections, adjacent to those used for phosphor screen autoradiography, were coated with a liquid photographic emulsion following our previously published protocol [5 (link), 9 (link), 22 (link), 34 (link)]. Immunohistochemistry was then performed on the nuclear emulsion-dipped sections. First the sections were washed for 5 min with PBS, then incubated with 2.5% normal horse blocking serum for 20 min, followed by the appropriate primary antibody - anti-tau PHF-1 (1:100, mouse, kind gift of Dr. Peter Davies), anti-Aβ (1:500, mouse, clone 6F/3D, Dako), anti α-synuclein (1:100, mouse, Zymed) or anti-phospho TDP-43 (pS409/410) (1:3000, mouse, Cosmo Bio CO) - for 40 min at 37 °C, washed with PBS twice for 2 min, and then incubated with the secondary antibody (ImmPRESS™ anti-mouse IgG (Vector Laboratories product MP-2400, Burlingame, CA) or ImmPRESS™ anti-rabbit Ig (Vector Laboratories product MP-7401, Burlingame, CA)) for 40 min at 37 °C. The sections were washed again with PBS twice for 2 min, and developed with DAB solution (Vector Laboratories product SK-4100). H&E was used for counterstaining. Photomicrographs were obtained on an upright Olympus BX51 (Olympus, Denmark) microscope using visible light.

To gain resolution at the cellular level, adjacent slides to those used in the phosphor screen autoradiography experiments, were dipped in a liquid photographic emulsion following our previously published protocols[1 (link), 29 (link), 31 (link)], followed by immunohistochemistry using the appropriate primary and secondary antibodies (primary antibodies used were: anti-tau PHF-1 (1:100, mouse, kind gift of Dr. Peter Davies), anti-Aβ (1:500, mouse, clone 6F/3D, Dako), anti α-synuclein (1:100, mouse, Zymed) and anti-phospho TDP-43 (pS409/410) (1:3000, mouse, Cosmo Bio CO); secondary antibodies used were ImmPRESS™ anti-mouse IgG (Vector Laboratories product MP-2400, Burlingame, CA) or ImmPRESS™ anti-rabbit Ig (Vector Laboratories product MP-7401, Burlingame, CA)) and developed with DAB solution (Vector Laboratories product SK-4100). H&E was used for counterstaining. Photomicrographs were obtained on an upright Olympus BX51 (Olympus, Denmark) microscope using visible light.

Formalin fixed, paraffin-embedded tissue blocks from neocortical areas, basal ganglia, thalamus, hippocampus, brainstem and cerebellum were evaluated. In addition to Hematoxylin and Eosin staining, the following monoclonal (mouse) antibodies were used for immunohistochemistry: monoclonal anti-p62 (1:1,000, BD Transduction, Lexington KY, USA), anti-tau AT8 (pS202/pT205, 1:200, Pierce Biotechnology, Rockford, IL, USA), anti-phospho-TDP-43 (pS409/410, 1:2,000, Cosmo Bio, Tokyo, Japan), anti-α-synuclein (1:2,000, clone 5G4, Roboscreen, Leipzig, Germany; specific for disease-associated form), anti-Aβ (1:50, clone 6F/3D, Dako, Glostrup, Denmark). The DAKO EnVision© detection kit, peroxidase/DAB, rabbit/mouse (Dako, Glostrup, Denmark) was used for visualization of the antibody reactions.