Image gauge 4

Using the software ImageGauge 4.0 (Fujifilm) and a tritium micro scale standard slide (ART 0123-1EA, American Radiolabeled Chemicals, Inc., St. Louis, MO, United States), specific binding (total – non-specific binding) was determined for striatum (Figure 2) of three replicate slices. Specific binding levels for doses of CP94253 were expressed as a percentage of vehicle-treated rats. These percentages were subtracted from 100 to obtain percent receptor occupancy.

In vitro autoradiography was performed on 14 postmortem brain sections (seven AD patients with a diagnosis confirmed by pathological assessment, and seven age-matched healthy controls) as previously described [17 (link)]. In brief, baseline and R-(–)-deprenyl hydrochloride competition experiments were performed on adjacent brain sections (20 μm thickness). Prepared frozen brain tissues were warmed to room temperature, air-dried, and pre-incubated in a buffer saline solution (138 mM NaCl and 27 mM KCl, pH 7.4 adjusted by NaOH) and 1% bovine serum albumin for 20 min. Brain tissues were then air-dried and incubated with ¹⁸F-THK5351 (1.85 μCi) and the R-(–)-deprenyl hydrochloride challenge was performed at concentrations of 150 nM and 500 nM for 150 min. After the incubation, brain tissues were dipped three times in the buffer solution for 3 min each time, once in 4 °C water for 30 s, and dried under a stream of cool air. The brain sections were subsequently exposed to a radioluminographic imaging plate (Fujifilm BAS-MS2025) for 20 min and obtained using FUJIFILM BAS-5000. Activity in photostimulated luminescence units per mm² was measured using Image Gauge 4.0 (Fujifilm) and the percentage of ¹⁸F-THK5351 total binding after R-(–)-deprenyl hydrochloride challenge was calculated.

Endothelial (bEnd.3) cell homogenate 8–12% was separated in SDS-PAGE gels, transferred to a polyvinylidene difluoride (PVDF) membrane, and incubated with primary antibodies against claudin-5 (1:1000; Invitrogen), occludin (1:1000; Invitrogen), phospho-IkB (1:1000; Cell Signaling), MMP2/9 (1;1000; Abcam, Cambridge, MA), and β-actin (1:3000; Sigma). Enhanced chemiluminescence (Denville Scientific, Metuchen, NJ) was used for visualization. Intensity of the bands was measured using Image Gauge 4.0 (FUJI Film, Tokyo, Japan).