Biomax ms

³²P-labeled and unlabeled mRNA probes were made in vitro using Riboprobe In Vitro Transcription Systems (Promega, Southampton, UK) in the presence or absence of 40 μCi of [α-³²P]rUTP (GE Healthcare) respectively. The BCL2 ARE probe was generated using the plasmidPCRII/bcl-2 ARE [30] (link). The resulting probe was separated from unincorporated nucleotides using ProbeQuant G-50 Micro Columns (GE Healthcare). E.coli-expressed purified ZFP36L1 protein (10–100 ng) or cell protein cell lysates (30 µg) were incubated with 50 000 to 100 000 cpm of α-³²P-labeled RNA probes, corresponding to approximately 30–100 fmol RNA. The proteins were incubated with RNA probes for 20 min on ice in RNA-binding buffer containing 20 mM HEPES (pH 7.6), 3 mM MgCl2, 40 mM KCl, 2 mM DTT, and 5% Glycerol in a total volume of 20 µl. RNase T1 and heparan sulfate (Sigma) were added to final concentrations of 50 U/ml and 5 mg/ml, respectively, and incubated on ice for another 20 min. RNA-protein complexes were resolved by electrophoresis (150 V for 3 hours at 4°C) on a 4% nondenaturing polyacrylamide gel using a Hoefer SE600 electrophoresis unit (Hoefer Scientific, Holliston, MA, USA). The gel was dried on a gel dryer (Hoefer SE1160 Gel Dryer), exposed to X-ray film (Kodak BioMax MS) and developed.

Fourteen micron sagittal sections (lateral 2.90 mm, bregma 0.48 mm, interaural 9.48 mm) of frozen rat brain hemispheres were collected on glass slides (3 sections/slide) and subjected to pre-hybridization treatment using an established ISHH protocol as previously described^{44 (link)}. Oligodeoxyribonucleotide cDNA probes complementary to: GluN1 (bases 746–780, NM008169.1), GluN2A (bases 1642–1676, NM008170.2) or GluN2B (bases 1758–1792, NM010350.2) were 3′-end labelled with [³⁵S]-dATP using terminal deoxynucleotidyl transferase (Promega, UK). Probes were then diluted in hybridization buffer, pipetted onto the tissue sections (1 × 10⁶ cpm/section), cover-slipped and then incubated for over 16 h at 34 °C in lidded Perspex trays lined with filter paper soaked with 4× SSC/50% formamide. Post-hybridization washes included 2× SSC rinse at room temperature to remove cover-slips, 3× in 0.5× SSC for 20 min at 55 °C, and 2× in 0.5× SSC for 30 min at room temperature. Slides were rinsed in ddH₂O, dried and exposed to X-ray film (Kodak, Biomax MS) for 7–28 days with ¹⁴C-microscales. Average grey densities over the frontal cortex, and dentate gyrus, CA1 and CA3 subfields of the hippocampus in the three sections from each group were measured using computer-assisted image analysis. Densities were calibrated to ³⁵S nCi/g tissue equivalents using commercial ¹⁴C-microscales.