In vitro transcription kit

tRNA^Gln was uniformly ³²P-labeled with α-³²P UTP (PerkinElmer, Japan, 3,000 Ci/mmol) using an in vitro transcription kit (Promega, Japan) and then gel-purified. The ³²P-labeled tRNA^Gln (10 000 cpm) was incubated in 10 μl of a solution containing 50 mM Tris–Cl, pH 7.4, 10 mM MgCl₂, 50 mM NaCl, 10 mM β-mercaptoethanol, 10% (v/v) glycerol and various amounts of CdiA–CT^EC869_H281A, Tu, or single-chain Tu-Ts (sg–Tu–Ts), at 37°C for 10 min. The solutions were then cooled on ice. The solutions were separated by 6% (w/v) native acrylamide gel electrophoresis (1 × TBE) at room temperature (∼25°C), as described (39 (link)). ³²P-labeled bands were quantified using a BAS-5000 imager (FujiFilm, Japan).

Specific riboprobes were generated from human TrkB-TK+ and TrkB-TK- containing plasmids as previously described [13 (link)]. Antisense riboprobes and sense strand RNAs were generated from linearized plasmid using SP6 and T7 polymerases respectively and an in vitro transcription kit as recommended by the manufacturer (Promega, WI, USA). [³⁵S] antisense and sense riboprobes were labelled to a specific activity of ~2 × 10⁹ cpm/μg by addition of radiolabelled UTP and were purified by ethanol precipitation. In situ hybridisation histochemistry was performed on 14-μm coronal sections containing the hippocampus as previously described [29 (link)]. Slides were exposed to film for one week or dipped in photographic emulsion (Kodak, type II NTB) for 12 weeks and then developed and counterstained with thionine. All sections were assayed together to limit interassay variability.

Sections from control and MIA male offspring were investigated for related mRNAs. Riboprobes (Table 2) were generated with ³⁵S-UTP (PerkinElmer) using an in vitro transcription kit (Promega, Madison, WI, USA). In situ hybridization was performed as previously described (44 (link)), using 5 ng/mL radiolabeled riboprobes in hybridization buffer, and ³⁵S-UTP labeled sense riboprobes as a negative control. Slides were exposed to BioMax MR (Kodak, Rochester, NY, USA) autoradiographic film (details in Table 2) alongside a ¹⁴C standard slide (American Radiolabeled Chemicals, St. Louis, MO, USA). Quantification of mRNAs was completed as mentioned above (see Quantification of Autoradiographic Images) with the standard Rodbard curve from the ¹⁴C standard slide.