Maxiscript transcription kit

Serial coronal sections (12 μm thick) were cut from 31 whole zebra finch brains using a cryostat, mounted on positively charged slides (SuperfrostPlus^®, Fisher Scientific, Pittsburgh, PA, United States), dried, and stored at −80°C until use. The brain sections were collected in such a way that consecutive sections were mounted on 18 parallel slides. For in situ hybridisation, [³⁵S]UTP-labelled riboprobes were generated from the DNA probes using a MAXIscript transcription kit (Ambion, Austin, TX, United States).
The preparation of tissue was performed using an mRNAlocator Kit (Ambion), according to the manufacturer’s instructions. Tissue was prepared using an mRNA-locator Kit (Ambion) according to manufacturer’s instructions. For hybridisation, we used 80 μl hybridisation buffer and 1 million DPM of labelled probe per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH = 7.4) at room temperature, and then at 65°C. After drying, slides were dipped in NTB nuclear track emulsion (Eastman Kodak, Rochester, NY, United States), stored for 3 weeks at 4°C for autoradiography, developed with Kodak Dektol developer, fixed with Kodak fixer, counterstained with Giemsa, and coverslipped with Cytoseal 60 (Stephens Scientific, Riverdale, NJ, United States).

Detection of sRNAs in Northern blot experiments was carried out as described [5 (link), 35 (link)]. sRNAs (25–35 μg) were separated by electrophoresis on 15% TBE-Urea gels (Invitrogen), electrotransferred to Hybond N1 filters at 400 mA for 1 h in 0.5X TBE, and cross-linked by ultraviolet irradiation (2 pulses at 1.2x10⁵ μJ per cm²). Ultrasensitive hybridization buffer UltraHyb (Ambion) was used for prehybridization and hybridization. Antisense-specific riboprobes were prepared by in vitro transcription (MAXIscript transcription kit; Ambion) and labeled with [α-32P] dUTP following supplier-recommended protocols. Riboprobes were treated as described previously [7 (link)] to result in an average size of 50 nucleotides (nt). An antisense-specific probe for the fkbA gene was amplified from genomic DNA with primers JOHE23654 and JOHE23559 and in vitro transcribed from the T7 promotor contained within the JOHE23654 primer sequence (S2 Table). This in vitro transcribed probe was also used to detect antisense fkbA mRNA in the different strains by Northern blot (S10 Fig). The carB antisense-specific riboprobe was obtained from the in vitro transcription of linearized plasmid pMAT652 [24 (link)]. JOHE37682 and JOHE37683 were used to amplify the 5S rRNA from genomic DNA and in vitro transcribed from the T7 promotor contained in both primer sequences (S2 Table).

Two freshly frozen DMPFC brain blocks of subjects were used: one from a 75-year-old female control subject with negative clinical reports for any major diseases and one from a 72-year-old female suicidal individual. Using a cryostat, serial coronal sections (12 μm) were cut and mounted on positively charged slides (SuperfrostPlus^®, Fisher Scientific), dried and stored at −80 °C until use. Further steps were performed according to the procedure described previously by Dobolyi et al. [153 (link)]. Briefly, antisense [35S]UTP-labeled riboprobes were generated from the above-described DNA probes using T7 RNA polymerase of the MAXIscript Transcription Kit (Ambion, Austin, TX, USA) and used for hybridization at 1 million DPM (discharges per minute) activity per slide. Washing procedures included a 30 min incubation in RNase A, followed by decreasing concentrations of sodium-citrate buffer (pH 7.4) at room temperature and subsequently at 65 °C. Following hybridization and washes, slides were dipped in NTB nuclear track emulsion (Eastman Kodak) and stored at 4 °C for 3 weeks for autoradiography. Then, the slides were developed and fixed with Kodak Dektol developer and Kodak fixer, respectively, counterstained with Giemsa and coverslipped with Cytoseal 60 (Stephens Scientific, Kalamazoo, MI, USA).