TUNEL staining was carried out as described [57 (link)]. Morpholinos-injected albinos embryos fixed in MEMFA were rehydrated in PBT and washed in TdT buffer (Invitrogen) for 30 min. End labelling was carried out overnight at room temperature in TdT buffer containing 0.5 μm DIG-dUTP and 150 U/ml TdT (Invitrogen). Embryos were then washed for 2 h at 65°C in PBS/1 mM EDTA. DIG was detected with anti-DIG Fab fragments conjugated to alkaline phosphatase (Roche, Indianapolis, Indiana, USA; 1:2,000) and the chromogenic reaction performed using BM purple (Roche, Indianapolis, Indiana, USA). Injection of a Morpholino against Sf3b4 was used as a positive control for induction of cell death [27 (link)]. For each MO, a subset of injected embryos injected was processed for in situ hybridization against sox10 as internal control for the effect of the MOs. TUNEL dots were counted in the neural crest region on each side. The neural crest region was defined as the lateral part of the anterior neural fold. Differences between injected and uninjected sides were plotted as well as the frequency distribution of TUNEL dots on the injected side for each condition.
Tdt buffer
Manufactured by Thermo Fisher Scientific
Sourced in United States
TdT buffer is a reagent used in molecular biology applications. It is designed to maintain the optimal pH and ionic conditions for the enzymatic activity of the Terminal deoxynucleotidyl Transferase (TdT) enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Tdt enzyme, by Thermo Fisher Scientific (3 mentions)
Nbt bcip, by Roche (2 mentions)
Bm purple, by Roche (2 mentions)
Dig dutp, by Thermo Fisher Scientific (2 mentions)
Anti dig fab fragments conjugated to alkaline phosphatase, by Roche (2 mentions)
Qiaquick nucleotide removal kit, by Qiagen (1 mentions)
Yeast poly a polymerase, by Thermo Fisher Scientific (1 mentions)
Optical microscope, by Leica (1 mentions)
Biotin dutp, by Thermo Fisher Scientific (1 mentions)
Biotinylated 16 dutp, by Roche (1 mentions)
7 protocols using tdt buffer
1
TUNEL Staining for Detecting Cell Death
2
Fluorescent Labeling of RNA Precursor
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To label the 3΄-end of precursor 1 using Terminal Deoxynucleotidyl Transferase (TdT), DNA has to be attached to its 3΄-end because 3΄ terminal ribonucleotides are poorly accepted by the enzyme. Addition of a poly-deoxyadenosine (poly-dA) tail to the 3΄-end of precursor 1 was done in a final volume of 25 μl containing 60 pmol of precursor 1, 1× Poly(A) Polymerase reaction buffer (Affymetrix), 0.5 mM deoxyadenosine triphosphate (dATP) (Life Technologies) and 600 units of Yeast Poly(A) Polymerase (Affymetrix). The reaction was incubated at 37°C for 8 h and was column-purified using the QIAquick Nucleotide Removal Kit according to the manufacturer's instructions (Qiagen). The eluted oligonucleotide was ethanol precipitated and desalted by drop dialysis. The 3΄-end labeling of precursor 1 was done in a final volume of 30 μl containing 1× TdT buffer (Life Technologies), 100 μM fluorescein-12-uridine triphosphate (Fluorescein-12-UTP) (Roche Life Science) and 30 units of TdT (Life Technologies). The reaction was incubated at 37°C for 60 min and the 3΄-fluorescein-labeled precursor 1 was phenol/chloroform extracted, ethanol precipitated and gel-purified.
3
TUNEL Assay for Apoptosis Detection
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Fixed frozen brain sections were permeabilized in 0.1% Triton X-100 for 15 minutes. After washing in PBS, sections were incubated at 37°C for 1 hour in a 50-μL solution containing 1× TdT buffer (catalog No. 16314015; Life Technologies, Rockville, MD, USA), 11.1 μM biotin-dUTP (catalog No. B32766; Life Technologies), and 20 U of TdT (Life Technologies). The sections were blocked with 2% bovine serum albumin in PBS, incubated in streptavidin-horseradish peroxidase (1:4000; Haoran Biotech Co., Shanghai, China) for 30 minutes, and stained with 3,3′-diaminobenzidine (1:9; Beijing Zhongshan Jinqiao Co., Beijing, China) for 15 minutes. The coverslips were then mounted using mounting medium and examined under an optical microscope (Leica, Oskar-Barnack-Straße, Germany).
4
Detecting Apoptosis in Xenopus Embryos
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TUNEL assay of Xenopus embryos was performed according to the Harland protocol (Conlon lab) available in Xenbase. Stored embryos were rehydrated and washed in 1xPBS and incubated for 1 hour at RT in TdT buffer (Invitrogen). Then, 150 U/ml TdT enzyme (Invitrogen) and 0.1 µl of DdUTP (Roche) per 100 µl buffer were added to the buffer solution and the embryos were incubated overnight at RT. The next day, embryos were washed 2x1 hour at 65°C in 1 mM EDTA/PBS and in 1xPBS 4x1 hour at RT, followed by 2–10 min washes in 1xMAB. Then they were blocked in 2%BMB blocking solution for 1 hour at RT and incubated in a 1/3000 dilution of anti-digoxigenin AP antibody in BMB block for 4 hours RT or overnight at 4°C. Antibody was washed away by 5x1 hour washes in MAB. Endogenous phosphatases were blocked by 2x10 min washes in alkaline phosphatase buffer and then NBT/BCIP (Roche) was added to the embryos. Chromogenic reaction was stopped by a quick wash in 1XMAB and then the embryos were fixed overnight in 1xMEMFA at RT. The next day embryos were imaged after clearing in two parts Benzyl Benzoate and one part Benzyl Alcohol after dehydration (Murray's Clearing Medium) (2∶1 BB: BA).
5
Spatial Distribution of Apoptotic Cells After Ischemia
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To clarify the spatial distribution of apoptotic cells after cerebral ischemia, we performed terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) staining as previously described (n = 8 each) [1 (link)]. Fixed sections were incubated with NeuroPore (Trevigen) for 30 min. They were placed in 1 × terminal deoxynucleotidyl transferase (TdT) buffer (Invitrogen) with a TdT enzyme (Invitrogen) and biotinylated 16-dUTP (Roche Diagnostics) at 37 °C for 90 min. The avidin–biotin technique was applied and then the nuclei were counterstained with methyl green solution for 2 min. For more precise confirmation about SR-FLIVO-FMNP probe tracking within the apoptotic cells, high resolution transmission electron microscopy (HRTEM) was recorded for the cells located at ischemic and non-ischemic lesions.
6
TUNEL Staining of Morpholino-Induced Cell Death
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TUNEL staining was carried out as described (Hensey and Gautier, 1998) . Morpholinos-injected albinos embryos fixed in MEMFA were rehydrated in PBT and washed in TdT buffer (Invotrogen) for 30 min. End labeling was carried out overnight at room temperature in TdT buffer containing 0.5 μM DIG-dUTP and 150 U/ml TdT (Invitrogen). Embryos were then washed for 2 hours at 65°C in PBS/1 mM EDTA. DIG was detected with anti-DIG Fab fragments conjugated to alkaline phosphatase (Roche, Indianapolis IN; 1:2000) , and the chromogenic reaction performed using BM purple (Roche, Indianapolis). Injection of a Morpholino against Sf3b4 was used as a positive control for induction of cell death (Devotta et al., 2016) . For each MO, a subset of injected embryos injected was processed for in situ hybridization against sox10 as internal control for the effect of the MOs. TUNEL dots were counted in the neural crest region on each side. The neural crest region was defined as the lateral part of the anterior neural fold.
Differences between injected and uninjected sides were plotted as well as the frequency distribution of TUNEL dots on the injected side for each condition.
Differences between injected and uninjected sides were plotted as well as the frequency distribution of TUNEL dots on the injected side for each condition.
7
TUNEL Assay for Xenopus Embryos
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TUNEL assay of Xenopus embryos was performed according to the Harland protocol (Conlon lab) available in Xenbase. Stored embryos were rehydrated and washed in 1xPBS and incubated for 1 hour at RT in TdT buffer (Invitrogen). Then, 150 U/ml TdT enzyme (Invitrogen) and 0.1 ml of DdUTP (Roche) per 100 ml buffer were added to the buffer solution and the embryos were incubated overnight at RT. The next day, embryos were washed 2x1 hour at 65uC in 1 mM EDTA/PBS and in 1xPBS 4x1 hour at RT, followed by 2-10 min washes in 1xMAB. Then they were blocked in 2%BMB blocking solution for 1 hour at RT and incubated in a 1/3000 dilution of anti-digoxigenin AP antibody in BMB block for 4 hours RT or overnight at 4uC. Antibody was washed away by 5x1 hour washes in MAB. Endogenous phosphatases were blocked by 2x10 min washes in alkaline phosphatase buffer and then NBT/BCIP (Roche) was added to the embryos. Chromogenic reaction was stopped by a quick wash in 1XMAB and then the embryos were fixed overnight in 1xMEMFA at RT. The next day embryos were imaged after clearing in two parts Benzyl Benzoate and one part Benzyl Alcohol after dehydration (Murray's Clearing Medium) (2:1 BB: BA).
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