Bas ms 2025

An aliquot of the McsB kinase (1 µM) was incubated with 12 µM substrate at 37°C in a buffer containing 50 mM HEPES-NaOH pH 7.5, 50 mM KCl, 2% (v/v) glycerol, 10 mM β-mercaptoethanol, 7.5 mM MgCl₂, 5 mM cold ATP, and 50 µCi γ−32^P ATP (Hartmann Analytics). Reactions were quenched at given timepoints upon addition of 5x SDS sample buffer and resolved on a Tris-glycine SDS-PAGE. Gels were dried, exposed to a phosphor imaging plate (BAS-MS 2025, Fuji film) overnight and visualized with a Typhoon Biomolecular imager (GE Healthcare). Quantification was performed by measuring the integrated pixel density of each band using Adobe Photoshop CC.

Tissue sections were placed into a BAS-Cassette (2325, Fujifilm) and exposed to a phosphor imaging plate (BAS-MS 2025, Fujifilm) for 15 h at room temperature. The images were developed on a BAS-5000 reader (Fuji Photo Film Co., Ltd.) and analyzed with Adaptive Image Deconvolution Algorithm (AIDA) Image Analyzer v.450 software.

Neutral lipids were extracted according to the Bligh and Dyer method^{43 (link)} and resuspended in chloroform. Each extract was seeded in 1 cm lanes on a 13 × 15 cm silica-aluminum plate (Merck). The mobile phase consisted of a mixture of acetic acid:diethyl ether:hexane (1:20:80 v/v/v). The plate was left to air dry and was exposed on a photosensitive screen overnight (Imaging Plate BAS-MS 2025, 20 × 25 cm, Fujifilm). Autoradiography scanning was performed on Typhoon FLA 7000 (GE Life Biosciences). For better visualization, minor adjustments in brightness and contrast were performed on the entire image using the Fiji software (ImageJ)^{44 (link)}. For radioactivity measurement, each spot was first visualized with a sublimated iodine spray, then scraped and directly resuspended in scintillation liquid. Full-length versions of the autoradiograms are included in Supplementary Fig. S2 and S3.

Control rats and rats treated with CCl₄ for 6 weeks were injected intravenously with [¹⁸F]FEDAC and then sacrificed after 1 h. Liver lobules were quickly removed and frozen with dry-ice powder. Frozen liver sections (10-μm-thick) were prepared using a microtome and thaw-mounted on glass slides. The sections were exposed to the imaging plate (BAS-MS2025; FUJIFILM, Tokyo) for 1 h. Radioactivity in the liver sections was detected by scanning the imaging plate using a BAS-5000 system (FUJIFILM). The liver sections after autoradiography were used for immunohistochemical staining with TSPO antibody.
The sections were incubated with a rabbit anti-mouse TSPO primary antibody overnight at 4 °C. After washing with PBS, the sections were then incubated with biotin-conjugated goat anti-rabbit IgG secondary antibody (1:1000; Invitrogen) for 1 h at room temperature, followed by tyramide signal amplification using a Fluorescein System (PerkinElmer). Fluorescence in the liver sections was detected using a FLA-5100 Fluoro-Imageanalyzer system (FUJIFILM).

Overall, 2–5 μg of total sRNAs were run on 18% denaturing polyacrylamide gel and transferred to a positively charged nylon membrane (GE Healthcare Amersham Hybond N+) on a Trans-Blot SD Semi-Dry Transfer Cell (Biorad). The RNA was ultraviolet-cross-linked to the membrane with Spectrolinker XL-1500 (Spectroline, ‘optimal crosslink'). Prehybridization was performed with Church Buffer (0.5 M NaH₂PO₄/Na₂HPO₄ pH 7.2, 1 mM EDTA, 7% SDS) at 37 °C overnight. 10 pmol of DNA probes (Supplementary Table 4) were labelled with T4 PNK (NEB) and 10 pmol [γ-³²P]-ATP (Hartmann Analytic) at 37 °C for 60 min. The labelled probes were purified with an Illustra MicroSpin G-25 column (GE Healthcare), mixed with 5 ml Church Buffer, and incubated with the membrane for 5 h at 37 °C. The membrane was rinsed once with 2x SSC buffer (0.3 M NaCl, 0.03 M sodium citrate, pH 7) and then washed three times with 2x SSC buffer for 15 min at 37 °C. The membrane was wrapped in cling film and exposed to a storage phosphor screen (BAS MS 2025—Fujifilm Corporation) overnight up to 2 days at −80 °C. The screen was scanned with a Typhoon FLA 9500 (GE Healthcare). For a second hybridization of the same membrane, the membrane was stripped in boiling 0.1% SDS for 5 min and subsequently prehybridized.

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.