Cellular-Level Autoradiography and Immunohistochemistry

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.