Amersham typhoon phosphorimager

Fresh-frozen brains were cryosectioned (Leica CM3050S; 10 µm), thaw-mounted on glass slides (Fisher Superfrost Plus Gold) and stored at −80°C for later use. [³H]PBR-28 ARG was carried out to evaluate specific binding, where tissues were incubated with 3 nM [³H]PBR-28 with a 10 µM unlabelled PK-11195 or a vehicle in 50 mM Tris/0.9% NaCl pH 7.4 for 1 h at room temperature. [³H]L-deprenyl ARG was carried out to evaluate specific binding, where tissues were incubated with 4 nM [³H]L-deprenyl with a 10 µM unlabelled lazabemide hydrochloride or a vehicle in PBS + 0.1% BSA for 1 h at room temperature. Tissue sections were subsequently washed with saline (2 × 5 min, 4°C) and deionized water (1 × 10 s, 4°C). Slides were air-dried and exposed to phosphor screens (BAS-IP TR4020; GE Healthcare) for 4 days with a full range tritium standard to permit quantification of radioligand binding (American Radiolabeled Chemicals, Inc.; St Louis, MO). Images were generated by scanning with an Amersham Typhoon phosphorimager (GE Healthcare). Region of interest (ROI) analysis was performed using MCID 7.0 imaging suite (Interfocus Imaging, Cambridge, UK). Raw nanocurie/milligram (nCi/mg), per cent specific binding {% specific binding = [(total signal − non-specific signal)/(total signal)] × 100} is reported.

Total RNA was extracted and prepared as described previously^{58 (link)}. For Northern blot analysis, RNA samples were separated on 6% polyacrylamide/7 M urea gels and transferred to Hybond-XL membranes (GE Healthcare). Membranes were hybridized in Roti-Hybri-Quick buffer (Roth) at 42 °C with [³²P] end-labeled DNA oligonucleotides. Oligonucleotides used for probing are listed in Supplementary Table S4. Membranes were washed in three subsequent steps with SSC (5x, 1x, 0.5x)/0.1% SDS wash buffer. Signals were visualized on a Amersham Typhoon phosphorimager (GE Healthcare) and quantified with GelQuant software (BiochemLabSolutions).

Genomic DNA was prepared by resuspending cell pellets in equal volumes of DNA buffer (100 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 100 mM NaCl, 1% SDS) and Phenol:chloroform:isoamyl alcohol (25:24:1) saturated with 10 mM Tris (pH 8.0) and 1 mM EDTA, and lysing cells with glass beads using FastPrep (MP Biomedicals). Eight hundred nanogram genomic DNA was digested overnight at 37 °C, separated on 1% agarose gel, transferred to Hybond-XL (GE Healthcare), probed with ³²P labeled telomeric DNA probe, and visualized with Amersham Typhoon Phosphorimager (GE Healthcare)^{34 (link),59 (link)}.

The samples were lysed in BN PAGE sample buffer (Thermo Fisher Catalog# BN2008) containing 1% digitonin and protease inhibitors following the manufacturer’s recommendation. The lysates were treated with benzonase at room temperature for 30 minutes to shear the DNA, and cleared by centrifugation for 15 mins. Prior to electrophoresis, the samples were mixed with Coomassie G-250 and resolved in 3–12% NativePAGE Bis-Tris gel (Invitrogen Catalog #BN1003BOX) as per the manufacturer’s recommendations. The gels were then transferred on PVDF membranes (Immunobilon-FL, Millipore) following the manufacturer’s recommendations. After transfer, the membranes were incubated in 8% acetic acid for 15 minutes to fix the proteins, rinsed with deionized water, and air-dried. The lane containing the size standard was cut from the membrane and stained with Ponceau S, and imaged on Amersham Typhoon Phosphorimager (GE Healthcare) using the Cy2 filter set. The membranes were blocked, probed with antibodies, and imaged as described above in the denaturing gel electrophoresis protocol.