Terminal deoxynucleotidyl transferase

Manufactured by Roche

Sourced in Germany, United Kingdom, Switzerland, United States

Terminal deoxynucleotidyl transferase is an enzyme that catalyzes the addition of deoxynucleotides to the 3' end of DNA molecules in a template-independent manner.

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Lab products found in correlation

35s thio datp, by Cytiva (5 mentions) Biotin 16 dutp, by Roche (4 mentions) Apotome 2, by Zeiss (3 mentions) Brij 35, by Merck Group (3 mentions) Bovine serum albumin (bsa), by Merck Group (3 mentions) Fluorescein dutp and terminal deoxynucleotidyltransferase, by Roche (2 mentions) Bca protein assay kit, by Thermo Fisher Scientific (2 mentions) Proteinase k, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Merck Group (2 mentions) Permount, by Thermo Fisher Scientific (2 mentions) Mightyamp dna polymerase, by Takara Bio (1 mentions) Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions) Lightcycler 480 sybr green 1 master, by Roche (1 mentions) Lightcycler 480 2 system, by Roche (1 mentions) βmax film, by Kodak (1 mentions) Qiaquick nucleotide removal kit, by Qiagen (1 mentions) 35 s datp, by PerkinElmer (1 mentions) Streptavidin alexa fluor 568 conjugate, by Thermo Fisher Scientific (1 mentions) Eclipse e600 epifluorescence microscope, by Nikon (1 mentions)

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27 protocols using terminal deoxynucleotidyl transferase

In Vitro Neuronal Apoptosis Detection

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After fixation, neuronal cultures were incubated with 1X Terminal Deoxynucleotidyl Transferase (TdT) Buffer (3 mM trizma base, 14 mM cacodylate sodium, 0.1 mM cobalt chloride, pH 7.2) for 15 minutes at 37°C and subsequently with TdT reaction mixture containing TdT buffer, 1mM Biotin-16-dUTP, 400 U/mL Terminal Deoxynucleotidyl Transferase [Roche, Indianapolis, IN] for 1 hour at 37°C. After incubation, the enzyme activity was blocked by washing with 2X Saline-sodium citrate (SSC). Unspecific binding sites were blocked by incubating for 30 minutes with a blocking solution containing 1% BSA and 0.1% Tx-100 at RT. Neurons were incubated with 1:3000 Streptavidin Alexa Fluor® 568 conjugate (ThermoFisher Scientific), for 1 hour at RT in the dark. After incubation, neurons were washed repeatedly with PBS and standard immunofluorescence protocol for NeuN antibody and DAPI staining was carried out.
Pictures were taken using Nikon Eclipse E600 epi-fluorescence microscope (Nikon Corp, Tokyo, Japan) with 20× objective. Cell counting was done using ImageJ software (National Institute of Health).

Colla E., Panattoni G., Ricci A., Rizzi C., Rota L., Carucci N., Valvano V., Gobbo F., Capsoni S., Lee M.K, & Cattaneo A. (2017). Toxic properties of microsome-associated alpha-synuclein species in mouse primary neurons.. Neurobiology of disease, 111, 36-47.

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Single-Cell Real-Time PCR Protocol

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The cDNA template for real-time PCR was prepared as previously reported (14 (link)) from FeSP neurons isolated and purified as described above. In brief, total RNA was purified from 50 neurons per sample using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and reverse transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and the oligo(dT) primer (5′-TATAGAATTCGCGGCCGCTCGCGATAATACGACTCACTATAGGGCG(T)₂₄-3′). After the addition of a poly(A) tail with terminal deoxynucleotidyl transferase (Roche Diagnostics, Basel, Switzerland), second-strand cDNA was synthesized using MightyAmp DNA polymerase (Takara Bio, Shiga, Japan) and the tagging primer (5′-TATAGAATTCGCGGCCGCTCGCGA(T)₂₄-3′). The cDNA was then amplified by 18 cycles of suppression PCR using MightyAmp DNA polymerase (Takara Bio) and the 5′-aminated primer (5′-NH₂-GTATAGAATTCGCGGCCGCTCGCGAT-3′).
Real-time PCR was performed on the LightCycler 480 System II using the LightCycler 480 SYBR Green I Master (Roche Diagnostics). Melt curve analysis was performed to verify that a single amplicon was obtained in each sample. Normalization was performed using the geometric mean of actb (GenBank accession number: NM_001104808) and eef1a (GenBank accession number: NM_001104662), according to Vandesompele et al. (47 ). The primers used for real-time PCR are listed in Table S3.

Fleming T., Tachizawa M., Nishiike Y., Koiwa A., Homan Y, & Okubo K. (2023). Estrogen-dependent expression and function of secretogranin 2a in female-specific peptidergic neurons. PNAS Nexus, 2(12), pgad413.

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In situ Hybridization of NPY Expression in Mouse Brain

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In situ hybridization was performed on brain sections from wild type and catNPY mice to examine NPY expression following procedures described previously [4] (link). Briefly, coronal brain sections (25 μm) were cut on a cryostat and thaw-mounted on Superfrost^® slides (Menzel-Glaser, Braunschweig, Germany). Matching sections from same coronal brain level were assayed together using DNA oligonucleotides complementary to mouse NPY (5′-GAGGGTCAGTCCACACAGCCCCATTCGCTTGTTACCTAGCAT-3′). The oligonucleotides were labelled with [35S] thio-dATP (Amersham Pharmacia) using terminal deoxynucleotidyltransferase (Roche, Mannheim, Germany) and hybridized with sections. Hybridization signals were visualized by exposing sections to βmax film (Kodak, Rochester, NY, USA) for 5–10 days. The film was then scanned to obtain the digital images of the hybridization signals on the sections.

Zhang L., Lee I.C., Enriquez R.F., Lau J., Vähätalo L.H., Baldock P.A., Savontaus E, & Herzog H. (2014). Stress- and diet-induced fat gain is controlled by NPY in catecholaminergic neurons. Molecular Metabolism, 3(5), 581-591.

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In Situ Hybridization of Tph2 mRNA

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Antisense oligoprobes complementary to rat and mouse tph2 mRNA (5′-TCC GTC CAA ATG TTG TCA GGT GGA TCC AGC CTC ACA ATG GTG GTC-3′; position 505; NM_173391) were synthesized by and purchased from Integrated DNA Technologies (Coralville, Iowa). The oligonucleotides were labeled with [35-S]dATP (Perkin Elmer, Waltham, Massachusetts) at the 3′ end using terminal deoxynucleotidyltransferase (Roche, Tucson, AZ) and purified using the QiaQuick Nucleotide Removal kit (Qiagen, Valencia, CA). Each slide was coverslipped with 110 μl of the hybridization buffer containing 0.1 M EDTA, SSC, single-stranded salmon sperm DNA, torula yeast tRNA, Denhardt's solution, formamide, dextran sulfate, and 1M DTT. Sections were incubated in a humidified chamber for 16 h at 42°C. Following hybridization, coverslips were removed and the slides were washed four times in 1X SSC buffer for 15 min at 55°C, once in 0.5X SSC buffer for 20 min at room temperature, and once again in 0.1X SSC buffer for 20 min at room temperature. Slides were then dipped in ddH₂O and air-dried. Slides were exposed to BioMax Kodak autoradiography film in a sealed film cassette in a dark room at room temperature for 4 days.

Hiroi R., Weyrich G., Koebele S.V., Mennenga S.E., Talboom J.S., Hewitt L.T., Lavery C.N., Mendoza P., Jordan A, & Bimonte-Nelson H.A. (2016). Benefits of Hormone Therapy Estrogens Depend on Estrogen Type: 17β-Estradiol and Conjugated Equine Estrogens Have Differential Effects on Cognitive, Anxiety-Like, and Depressive-Like Behaviors and Increase Tryptophan Hydroxylase-2 mRNA Levels in Dorsal Raphe Nucleus Subregions. Frontiers in Neuroscience, 10, 517.

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In situ hybridization analysis of hypothalamic gene expression

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Fresh frozen brains were sectioned at 30 μm thickness and thaw-mounted on Superfrost Plus® glass microscope slides (Lomb Scientific Pty Ltd., NSW 2229, Australia). In situ hybridisation was performed, as previously described38 (link). Briefly, matching hypothalamic sections of deletion and control mice were hybridised with candidate mRNAs, which were labelled with [³⁵S] thio-dATP (Amersham Pharmacia Biotech, Buckinghamshire, UK) using terminal deoxynucleotidyltransferase (Roche, Mannheim, Germany). Silver grain densities of labelled mRNAs were analysed and compared using ImageJ software (US National Institutes of Health).
DNA oligonucleotides used included those complementary to the mRNAs of mouse Snord116, mouse GHRH, mouse NPY, mouse POMC, mouse MCH, mouse orexin, mouse GnRH, mouse oxytocin and mouse TH (see Supplementary Table 2).

Qi Y., Purtell L., Fu M., Lee N.J., Aepler J., Zhang L., Loh K., Enriquez R.F., Baldock P.A., Zolotukhin S., Campbell L.V, & Herzog H. (2016). Snord116 is critical in the regulation of food intake and body weight. Scientific Reports, 6, 18614.

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Histological analysis of inguinal fat

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Inguinal fat fixed in 10% formalin was dehydrated through a graded ethanol series, embedded in paraffin, and cut into 5 μm sections. After staining with hematoxylin and eosin, sections were examined under a light microscope (OLYMPUS IX71) equipped with a SPOT RT color-2000 digital camera (Diagnostic Instruments, Sterling Heights, MI, USA). For uncoupling protein 1 (UCP1) immunostaining, sections were deparaffinized, rehydrated, and incubated with 0.5% Triton X-100, then blocked using 5% goat serum in phosphate-buffered saline (PBS). The primary antibody was rabbit antibody against human UCP1 (Abcam, Cambridge, UK), used at a dilution of 1:100 in PBS, whereas the secondary antibody was biotinylated goat anti-rabbit IgG antibody (Dako, Carpinteria, CA, USA), at a dilution of 1:250 in PBS. For detection of apoptosis, the TUNEL assay used terminal deoxynucleotidyl transferase and tetramethyl-rhodamine-dUTP (Roche Applied Science, Indianapolis, IN, USA) to directly label DNA strand breaks (red fluorescence). Images were acquired using a fluoromicroscope equipped with a SPOT RT color-2000 digital camera (Diagnostic Instruments, Sterling Heights, MI, USA).

Chang Y.Y., Su H.M., Chen S.H., Hsieh W.T., Chyuan J.H, & Chao P.M. (2016). Roles of Peroxisome Proliferator-Activated Receptor α in Bitter Melon Seed Oil-Corrected Lipid Disorders and Conversion of α-Eleostearic Acid into Rumenic Acid in C57BL/6J Mice. Nutrients, 8(12), 805.

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Apoptosis Detection in Tissue Sections

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The tissue sections were incubated with proteinase K, washed in phosphate-buffered saline, incubated with permeabilization solution, blocking buffer, and then washed two times with PBS. The sections were stained with terminal deoxynucleotidyl transferase and fluorescein isothiocyanate- dUTP (Roche Applied Science, Indianapolis, IN, USA) for 60 min at 37°C in order to detect the number of apoptotic nuclei. The tissue sections were also stained with 0.1 µg/mL 4’,6-diamidino-2-phenylindole (DAPI) for 5 min, and the nuclei were detected by UV light microscopy at 454 nm. Photomicrographs were obtained using a Zeiss Axiophot microscope. All counts were performed by at least three independent individuals in a blinded manner.

Chiang W.D., Huang C.Y., Paul C.R., Lee Z.Y, & Lin W.T. (2016). Lipolysis stimulating peptides of potato protein hydrolysate effectively suppresses high-fat-diet-induced hepatocyte apoptosis and fibrosis in aging rats. Food & Nutrition Research, 60, 10.3402/fnr.v60.31417.

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Blastocyst TUNEL Staining Protocol

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Blastocysts were washed three times in PBS-PVA then fixed in 3.7% paraformaldehyde (w/v) for 1 h at room temperature. After fixation, they were permeabilized with 0.5% Triton X-100 (v/v) for 1 h at 38.5°C. Permeabilized blastocysts were then incubated in the dark for 1 h at 37°C with fluorescein-conjugated deoxyuridine triphosphate (dUTP) and terminal deoxynucleotidyl transferase (Roche, Mannheim, Germany). After nick end labeling, the blastocysts were counterstained with 10 μg/mL Hoechst 33342 for 10 min at room temperature to label nuclei, washed in DPBS-PVA, mounted under a coverslip, and examined under a fluorescence microscope (TE300; Nikon).

Cai L., Jeong Y.W., Jin Y.X., Lee J.Y., Jeong Y.I., Hwang K.C., Hyun S.H, & Hwang W.S. (2020). Effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro culture on porcine pre-implantation embryos. PLoS ONE, 15(3), e0230247.

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TUNEL Assay for Apoptosis Detection

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Animals were sacrificed and immediately perfused via the right heart with 0.9% NaCl for 2 min followed by 10% formalin for 10 min. Organs, including the heart, lung, liver and kidney, were then removed and further fixed in 10% formalin for 48 h. TUNEL staining was performed with an In Situ Cell Death Detection Kit as instruction by the supplier. Briefly, the tissues were dehydrated, embedded in paraffin, and sectioned at a thickness of 5 µm. Sections were then dewaxed, rehydrated and incubated for 5 min in 0.1 M citrate buffer (pH 6.0) at 350 W in a microwave. After this, samples were immediately cooled in PBS and incubated with TMR coupled dUTP in the presence of terminal deoxynucleotidyl-transferase (Roche, Basel, Switzerland) at 37 °C for 60 min. Samples were embedded with mounting medium with DAPI prior to analysis. An excitation wavelength of 488 nm was used and evaluated using a ZEISS AXIO microscope (Carl ZEISS, Jena, Germany).

Li W., Wilson G.C., Bachmann M., Wang J., Mattarei A., Paradisi C., Edwards M.J., Szabo I., Gulbins E., Ahmad S.A, & Patel S.H. (2022). Inhibition of a Mitochondrial Potassium Channel in Combination with Gemcitabine and Abraxane Drastically Reduces Pancreatic Ductal Adenocarcinoma in an Immunocompetent Orthotopic Murine Model. Cancers, 14(11), 2618.

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In Situ Detection of DNA Fragmentation

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DNA fragmentation was detected in situ using TUNEL [24 ]. In brief, CMs were incubated with proteinase K, and DNA fragments were labeled with fluorescein-conjugated dUTP using terminal deoxynucleotidyl transferase (Roche Diagnostics). The total number of nuclei was determined by manual counting of DAPI-stained nuclei in 6 random fields per coverslip (original magnification, ×200). Digital photographs of fluorescence were acquired with a Zeiss microscope (ApoTome.2; Carl Zeiss) and processed with Adobe Photoshop CS5.1.

Bayoumi A.S., Park K.M., Wang Y., Teoh J.P., Aonuma T., Tang Y., Su H., Weintraub N.L, & Kim I.M. (2017). A carvedilol-responsive microRNA, miR-125b-5p protects the heart from acute myocardial infarction by repressing pro-apoptotic bak1 and klf13 in cardiomyocytes. Journal of molecular and cellular cardiology, 114, 72-82.

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