Everbrite mounting media

To visually characterize plaque and vascular binding of PiB in vitro, we used fixed tissue sections from the frontal cortex of two control cases (19 years and 51.3 years), DS (19.8 years), and DS with AD (DSAD) case (51.4 years) and the fluorescent cyano-PiB (CN-PiB). Sections were first mounted on slides and allowed to dry prior to incubation in 100 nM CN-PiB for 1 hour at RT using a similar protocol as published previously (Ikonomovic, et al., 2008 (link)) but at a lower concentration closer to the CN-PiB K_d to focus on high affinity imaging ligand-relevant binding. Slides were washed in PBS for 3× for 2 minutes then incubated briefly (30s) in TrueBlack™ lipofuscin autofluorescence quencher (Biotium, Hayward, CA). After three more 2-minute washes in PBS, slides were coverslipped using Everbrite™ mounting media (Biotium). Images were captured using an Olympus BX51 microscope with a Q Color 5 digital camera. A second set of sections was first incubated in CN-PiB as described above but prior to Trueblack quenching for autofluorescence, slides were incubated in 0.5% thioflavine-S (Sigma-Aldrich, St. Louis, MO) in 50% ethanol, differentiated in 50% ethanol, washed, and then exposed to TrueBlack. Sections were coverslipped using Everbrite™.

For tissue sample preparation of E12.5 embryos, the whole brain was dissected out in PBS and fixed in 2% paraformaldehyde (in 1X PBS) for 20–30min. For tissue sample preparation of older embryos, forebrain hemispheres were dissected out, part of the hippocampus was removed to expose the lateral ventricle, and the rest of the forebrain was fixed in 2% paraformaldehyde (in 1X PBS) for 25–30min. Fixed forebrain samples were cryoprotected with 20% sucrose (in 1X PBS), embedded in Tissue-Tek OCT, and cryosectioned at the thickness of 20–30μm. Sections were stored at −20°C before the immunostaining procedure. For immunostaining, both primary and secondary antibodies were diluted in 1xPBS containing 3% bovine serum albumin and 0.2% Triton-X-100. Antibody incubation steps (primary antibody: overnight; secondary antibody: 1–3h) were performed in a humidified chamber protected from direct light. Secondary antibodies with the following cyanine dyes were used: Cy2 (green), Cy3 (red), Cy5 (far red), and DyLight405 (blue). EverBrite^™ mounting media from Biotium (Fremont, CA) were used to mount coverslips and protect from photo bleaching.