Fresh-frozen brains (n=6/condition) were serially sectioned (at 10-μm thickness) throughout the forebrain using a cryostat, thaw-mounted onto Superfrost plus microslides (VWR, Batavia, IL), and stored at −20°C until hybridization. Semi-adjacent sections were processed for the in situ hybridization localization of GAD67 mRNA using a 35S-labeled cDNA probe, as previously described (Seroogy and Herman, 1997 ; Hemmerle et al., 2012 (link), 2015 (link); Makinson et al., 2015 (link)). Briefly, slides were pretreated, dehydrated and delipidated prior to hybridization. The hybridization probe was prepared from a linearized cDNA plasmid using T3 RNA polymerase and labeled with 35S-UTP (PerkinElmer, Boston, MA). The GAD67 plasmid (a generous gift from Dr. James Herman, University of Cincinnati) was contained in a Bluescript SK vector that consisted of 3086 bases (GenBank Gene ID: 24379). Sections were hybridized overnight, washed, treated with RNase, rinsed, air-dried, and exposed to BioMax MR film (Kodak, Rochester, NY) for 7 days. The films were developed with Kodak GPX developer and fixer.
35s utp
Manufactured by PerkinElmer
Sourced in United States
35S-UTP is a radioisotope-labeled nucleotide used in molecular biology applications. It serves as a radioactive tracer for the detection and quantification of RNA in various experimental techniques.
Automatically generated - may contain errors
Lab products found in correlation
Micro bio spin 30 column, by Bio-Rad (2 mentions)
Riboprobe system, by Promega (2 mentions)
In vitro transcription kit, by Promega (2 mentions)
Biomax mr, by Kodak (2 mentions)
Biomax mr film, by Kodak (1 mentions)
Superfrost plus micro slide, by Avantor (1 mentions)
Cm3050 cryostat, by Leica (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Topo ta cloning kit, by Thermo Fisher Scientific (1 mentions)
Gs 800, by Bio-Rad (1 mentions)
Maxi script labeling kit, by Thermo Fisher Scientific (1 mentions)
Taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Ntb nuclear track emulsion, by Kodak (1 mentions)
Autoradiographic film, by Kodak (1 mentions)
T7 polymerase, by Promega (1 mentions)
10 protocols using 35s utp
1
In Situ Hybridization of GAD67 mRNA in Brain
2
Radioactive Probe Synthesis for CRHR1, MR, GR
3
In Situ Hybridization Analysis of mRNAs
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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.
4
Ribonucleotide Probe Preparation
5
In Situ Hybridization for Circadian Clock Genes
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The Per1 probe corresponds to nucleotides 1 to 619 (GenBank accession number AF022992) and Per2 corresponds to nucleotides 229 to 768 of GenBank AF036893. PCR products had been cloned into pCR II TOPO vector using TOPO TA Cloning Kit (Life Technologies) (Oster et al., 2002 (link)). Linearization of vectors for in vitro transcription was done with EcoRI. 35S-UTP (PerkinElmer, Waltham, MA) labeled RNA probes were prepared using RNA Transcription Kit (Maxi Script Labeling Kit, Life Technologies) with T7 or T3 RNA polymerases according to the manufacturer's protocol. 10-µm cryosections were cut using a Leica CM3050 cryostat. Cryosections were fixed in 4% paraformaldehyde, acetylated in acetic anhydride and dehydrated with ethanol. Hybridization was performed over night at 55–58°C. Autoradiographs were analyzed by densitometry (Bio-Rad GS-800) using QuantityOne software (Bio-Rad). Three sections per brain were used and background values were calculated from adjacent tissue areas on the same slide for each section. Measurements from different animals/experiments were compared for statistical analysis using GraphPad Prism (GraphPad).
6
Expression Analysis of Ccdc88b Gene
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The expression of the Ccdc88b gene was monitored by whole mount in situ hybridization. PCR amplification of the designed Ccdc88b probe was performed using primers: 5′-GCGCTATAATACGACTCACTATAGGGAGATCCGAATCTTTGGACCTGCCTTCT-3′ and 3′-GCATTAATTTAGGTGACACTATAGAAGCGAAGCTAGCCGTATCCACTGCTTCA-5′, which contain embedded T7 and SP6 polymerases promoter sites (underlined). 35S-UTP labeled cRNA was synthesized in the presence of SP6 (antisense) and T7 (sense) RNA polymerases, and using 35S-UTP (>1,000 Ci/mmol; PerkinElmer). Tissues were fixed in 4% formaldehyde and hybridized with 35S-labeled cRNA antisense and sense probes. The final slides were dehydrated, and then dipped in Kodak NTB nuclear track emulsion, and exposed for 18 d. The slides were lightly counterstained with cresyl violet and analyzed under both light and dark field optics.
For reverse transcription PCR experiments, total spleen RNA was reverse transcribed into cDNA using oligo-d(T) primers and M-MuLV reverse transcription (Invitrogen). PCR amplification of exons 21–22 of Ccdc88b was performed using primers 5′-CCTGCAGGCTGAAAAGTCA-3′ and 5′-GCTCTCGTCGCTCTCATGGA-3′, and Taq DNA polymerase (Invitrogen), for 28 cycles at 30 s/94°C, 30 s/58°C and 30 s/72°C).
For reverse transcription PCR experiments, total spleen RNA was reverse transcribed into cDNA using oligo-d(T) primers and M-MuLV reverse transcription (Invitrogen). PCR amplification of exons 21–22 of Ccdc88b was performed using primers 5′-CCTGCAGGCTGAAAAGTCA-3′ and 5′-GCTCTCGTCGCTCTCATGGA-3′, and Taq DNA polymerase (Invitrogen), for 28 cycles at 30 s/94°C, 30 s/58°C and 30 s/72°C).
7
In Situ Hybridization of Circadian Genes
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Hypothalamic blocks for in situ hybridisation were cut into 20 μm sections using a cryostat, and thaw-mounted onto polylysine and gelatin coated slides. Radioactive cRNA riboprobes were prepared by plasmid linearisation and in vitro transcription reactions including 35S-UTP (Perkin-Elmer). Sections were hybridized overnight at 60°C with 5 x 105 cpm of probe per slide, subjected to Rnase-A digestion and stringency washes in sodium citrate buffer to remove non-specific probe hybridisation. Slides were then dehydrated in graded ethanol solutions and exposed to an autoradiographic film (Kodak). Exposure duration was optimized for each gene by repeat film exposures, depending on labeling intensity. Films were scanned on a 1640XL transmittance scanner (EpsonUK, Hemel Hempstead, Hertfordshire, UK) along with a calibrated optical density (OD) transmission step wedge (Stouffer, USA). Calibrated Integrated OD measurements of gene expression in the SCN were performed using ImageJ software. Homologous probes used in this paper correspond to the following nucleotide position of GenBank accession numbers: Avp nt 1–501 of NM_001126341, Vip nt 1–513 of NM_001126368, Grp nt 48–545 of NM_001009321, Per1 nt 1–456 of EF683160, Per2 nt 1–451 of EF583558, Bmal1 nt 1–836 of NM_001129734, Fbxl21 nt 342–709 of NM_001129738.
8
Radiolabelled Riboprobe In Situ Hybridization
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Riboprobes (Supplementary Table 1 ) were generated with 35S-UTP (CAT# NEG039H001MC Perkin Elmer, Waltham, Massachusetts, USA) using an in vitro transcription kit (CAT# P1121, Promega, Madison, Wisconsin, USA). In situ hybridisation was performed as previously described57 (link), using 5 ng/ml radiolabelled riboprobes in hybridisation buffer, and 35S-UTP labelled sense strand riboprobes as a negative control (Supplementary Figures 1 and 2 ). Slides were exposed to BioMax MR (Kodak, Rochester, NY, USA) autoradiographic film (Supplementary Table 1 ) alongside a 14C standard slide (American Radiolabelled Chemicals, St. Louis, MO, USA).
9
Liver cryosections for gene expression
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Cryosections (10 μm thick) were prepared from livers of 3-month-old WT mice, fixed in 4% formaldehyde, and hybridized as previously described [14] (link) with mouse sense (negative control) and antisense cRNA probes. The latter probes corresponded to coding regions of mouse PC7 (residues 1–213) or ApoF (full length), and were synthesized using 35S-UTP (PerkinElmer).
10
In situ Hybridization of c-fos mRNA
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A subset of representative rats from each group was used for in situ hybridization (IntA-5s and LgA-90s, n = 6/group; Sal-Ctrl, n = 10), to keep the hybridization procedure manageable. Self-administration data in this subset was comparable to that of the main groups (see Results). C-fos mRNA expression was labelled on 12-μm-thick coronal brain sections using a [ 35 S]-UTP-labelled riboprobe complementary to c-fos, as in (Bédard et al., 2011) . The complementary RNA probe for cfos mRNA was derived from 1.8 kb EcoRI fragment of a full-length rat c-fos cDNA. It was subcloned into pBluescript SK-1 plasmid and linearized with SmaI (Tremblay et al., 1999) . The probe was synthesized using a Promega riboprobe kit (Fisher Scientific, St-Laurent, QC), [ 35 S]-UTP (Perkin Elmer, Woodbridge, ON) and polymerase T7 (Promega, Fisher Scientific, St-Laurent, QC). Labelled brain slices were placed against Kodak Biomax MR X-ray film (VWR, Town of Mount-Royal, QC) for 4 days.
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