Whole-mount terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) was carried out as described (Hensey and Gautier, 1998 (link)) with minor modifications. Following fixation in MEMFA (100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7% (v/v) formaldehyde), embryos were rehydrated and permeabilized in PBS with 0.1% Tween-20, equilibrated in TdT buffer (Sigma) at room temperature (RT), and then incubated overnight in TdT buffer with digoxigenin-11 dUTP and TdT enzyme (Sigma). The reaction was stopped by incubating in 100 mM EDTA in PBS at 65 °C. An alkaline-phosphatase conjugated anti-dig antibody (Roche) was used with NBT/BCIP color reaction as in ISH.
Tdt buffer
Manufactured by Merck Group
Sourced in United States
TdT buffer is a solution used in molecular biology applications. It serves as a reaction buffer to facilitate the enzymatic activity of terminal deoxynucleotidyl transferase (TdT), an enzyme involved in DNA synthesis and modification processes.
Automatically generated - may contain errors
Lab products found in correlation
Protease k, by Merck Group (3 mentions)
Digoxigenin 11 dutp and tdt enzyme, by Merck Group (2 mentions)
Alkaline phosphatase conjugated anti dig antibody, by Roche (2 mentions)
Biotin dutp, by Roche (1 mentions)
Szx16 microscope, by Olympus (1 mentions)
Streptavidin conjugated horseradish peroxidase, by Roche (1 mentions)
Saponin, by Merck Group (1 mentions)
Anti digoxin and anti serum alkaline phosphatase complex, by Merck Group (1 mentions)
6 protocols using tdt buffer
1
Whole-mount TUNEL Assay for Apoptosis
2
Whole-mount TUNEL Assay for Apoptosis
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Whole-mount terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) was carried out as described (Hensey and Gautier, 1998 (link)) with minor modifications. Following fixation in MEMFA (100 mM MOPS pH 7.4, 2 mM EGTA, 1 mM MgSO4, 3.7% (v/v) formaldehyde), embryos were rehydrated and permeabilized in PBS with 0.1% Tween-20, equilibrated in TdT buffer (Sigma) at room temperature (RT), and then incubated overnight in TdT buffer with digoxigenin-11 dUTP and TdT enzyme (Sigma). The reaction was stopped by incubating in 100 mM EDTA in PBS at 65 °C. An alkaline-phosphatase conjugated anti-dig antibody (Roche) was used with NBT/BCIP color reaction as in ISH.
3
Apoptosis Evaluation via TUNEL Assay
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A TUNEL assay was performed by washing 4% paraformaldehyde-fixed cells on a coverslip once with PBS, followed by permeabilization using 0.5% saponin (Sigma-Aldrich) at room temperature for 30 min. Following a wash with terminal deoxynucleotidyl transferase (TdT) buffer (Roche Diagnostics, Indianapolis, IN, USA), cells were incubated with 0.5 µM biotin dUTP (Roche Diagnostics) and 150 U/ml of TdT (Sigma-Aldrich) in 30 µl of TdT buffer in a humidified chamber at 37°C for 30 min. Following two PBS washes, the cells were incubated with a 1/1,000 solution of streptavidin-conjugated horseradish peroxidase (Roche Diagnostics) in PBS for 10 min at room temperature. Coverslips were then washed for 30 min with three subsequent washes of PBS. Color was developed using TrueBlue peroxidase substrate (KPL, Inc., Gaithersburg, MD, USA), and coverslips were observed under an Olympus SZX16 microscope.
4
Quantifying Hippocampal Apoptosis in Rats
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Brain sections were fixed in 4% neutral PFA and taken out for paraffin embedding. Then, sections were dewaxed in xylene and hydrated in ethanol in gradient concentrations. Following rinsing sections in tap water, antigen retrieval was implemented at 80°C. Thereafter, the sections were incubated in presence of Protease K (Sigma, U.S.A.) and then placed in TDT buffer (Sigma, U.S.A.) for pre-incubation. After washed with PBS again, the sections were then incubated with anti-digoxin and anti-serum alkaline phosphatase complex (Sigma, U.S.A.) at 37°C overnight. Following washes in Tris-buffer, counter staining was performed with the corresponding reagent, and ended using Tris-buffer. Apoptosis in hippocampus of rats was observed under a microscope, and the results were quantified in Image-Pro Plus. Apoptotic ratio is calculated using the formula: Apoptotic ratio = TUNEL-positive cell quantity/total quantity of cells. The experiment was repeated three times.
5
Cardiac Apoptosis Quantification Assay
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Cardiac sections were implemented at 90°C, and then treated with Protease K (Sigma-Aldrich). Section were suspended in TDT buffer (Sigma-Aldrich), and incubated overnight with anti-serum alkaline phosphatase and anti-digoxin complex (Sigma-Aldrich). Following counter staining with DAPI, sections were observed under microscope. The apoptotic ratio was quantified using Image-Pro Plus.
6
Quantifying Hippocampal Apoptosis
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Hippocampal sections were implemented at 90°C for antigen retrieval, and treated with Protease K (Sigma-Aldrich). Sections were suspended in TDT buffer (Sigma-Aldrich), and incubated overnight with anti-serum alkaline phosphatase and anti-digoxin complex (Sigma-Aldrich). Following counterstaining with DAPI, sections were observed under microscope to determine apoptosis in hippocampus. The apoptotic ratio was quantified using Image-Pro Plus.
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