Following secondary antibody staining, tissues were washed thrice with 1X PBS-T for 10 minutes. Samples were then incubated in 0.1 M sodium citrate (Fisher Scientific) and 10% TritonX-100 (495μL of 0.1M Na-Citrate + 5μL of 10% TritonX-100 per sample tube) for 30 minutes in dark at 65°C. This step was performed to increase permeabilization and improve signal. Samples were then washed thrice with 1X PBS-T for 10 minutes. Next, the samples were incubated in the TUNEL dilution buffer (Roche Diagnostics, Catalog no. 11966006001) for 10 minutes at room temperature in the dark. Finally for the labelling step, samples were incubated for 2 hours at 37°C in 55μL TUNEL labelling solution [25μL Labeling Solution (Roche, Catalog no. 12156792910) + 25μL Dilution Buffer+5μL enzyme solution]. Samples were then washed thrice with 1X PBS-T for 10 minutes.
Tunel dilution buffer
Manufactured by Roche
Sourced in Germany, Japan
TUNEL Dilution Buffer is a laboratory reagent used to dilute and prepare samples for the TUNEL (Terminal deoxynucleotidyl transferase dUTP Nick End Labeling) assay. The buffer helps maintain the appropriate pH and ionic conditions for the TUNEL reaction.
Automatically generated - may contain errors
Lab products found in correlation
Tunel enzyme, by Roche (2 mentions)
Hoechst 33342, by Thermo Fisher Scientific (2 mentions)
Sodium citrate, by Thermo Fisher Scientific (1 mentions)
Blocking buffer, by Roche (1 mentions)
Tmr red, by Roche (1 mentions)
Vectashield mounting medium with 4 6 diamidino 2 phenylindole, by Vector Laboratories (1 mentions)
Biotin 16 dutp, by Roche (1 mentions)
Mowiol, by Merck Group (1 mentions)
In situ cell death detection kit, by Roche (1 mentions)
7 protocols using tunel dilution buffer
1
TUNEL Labeling for Apoptosis Detection
2
TUNEL Assay for Apoptosis Detection
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Wash the tissue in 1× PBST, thrice for 10 min each.
Prepare secondary antibody (anti-Rat Cy5) at a dilution of 1:300 in 1× PBST-NDS.
Incubate the tissue in a secondary antibody for 2 h in the dark at RT.
Wash the tissue in 1× PBST thrice for 10 min each.
Incubate in freshly prepared 495 μL of 100 mM Sodium Citrate stock solution + 5 μL of 10% Triton X-100 for 30 min in the dark at 65°C.
Wash the tissue in 1× PBST, thrice for 10 min each.
Incubate the tissue in ∼30 μL of TUNEL-Dilution Buffer (Roche) for 10 min in the dark at RT.
Incubate the tissue in a TUNEL labeling solution for 2 h at 37°C.
Wash the tissue in 1× PBST, thrice for 10 min each.
3
Apoptosis Induction in Osteosarcoma Cells
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SaOS-2 and MNNG/HOS cells seeded in a 4 well glass plate at a density of 5 × 103 cells/well were treated with or without CDDP (5 μg/ml), DOX (1 μg/ml), and/or OBP-301 (10 and 50 MOIs, respectively). In monotherapy, cells were treated with chemotherapy for 24 hours or infected with OBP-301 for 72 hours. In combination therapy, two days after viral infection with OBP-301, cells were further treated with chemotherapy for 24 hours. Cells were fixed in 1% paraformaldehyde in PBS at 4 °C for 10 minutes. Cells were stained by nick end-labelling method to detect the fragmented DNA, and Hoechst 33342 (Thermo Fisher Scientific Inc., Rockford, IL, USA) to observe the nuclear morphology of the cells. Briefly, cells were incubated with TdT reaction solution (400 U terminal transferase and 5 nmol Fluorescen-12-2′-deoxy-uridine-5′-triphosphate in 1.2 ml TUNEL Dilution Buffer (Roche Diagnostics, Mannheim, Germany)). The cells were then stained by Hoechst 33342 (20 μM) for 5 minutes at room temperature. The photographs of immunostained sections were obtained by fluorescent microscopy. The percentage of immunoreactive cells for TUNEL was calculated in five randomly selected fields in each group.
4
TUNEL Assay for Apoptotic Cell Detection
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Nuclei exhibiting apoptotic changes were identified by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay using the in situ cell death detection kit, TMRred (Roche Applied Science, Indianapolis, IN) according to the manufacturer’s recommendations. Briefly, the sections were rehydrated in PBS with 0.1% Tween 20 and then fixed in 4% paraformaldehyde at room temperature for 20 min. After several washes with T-PBS, the sections were fixed in 0.1% (w/v) sodium citrate (0.1% C5H3O7Na3·2H2O and 0.1% Triton X-100) at room temperature for 2 min. After washing, the sections were immunoreacted with anti-laminin antibody (1:500, Dako Japan, Tokyo, Japan) diluted in 1% (w/v) blocking reagent (Roche) in maleic acid buffer (0.1 mol/L maleic acid and 0.15 mol/L NaCl, pH 7.5) at room temperature for 1 h. After several washes in T-PBS, a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG secondary antibody (1:250, Sigma) in Blocking buffer (Roche) was applied for 1 h at room temperature. After several washes in T-PBS, the TUNEL reaction mix diluted in TUNEL Dilution Buffer (Roche) was applied and incubated at 37°C for 60 min in the dark. After several washes in T-PBS, the sections were cover slipped using Vectashield mounting medium with 4′, 6-diamidino-2- phenylindole (Vector Labs, Burlingame, CA).
5
TUNEL Staining in Paraffin-Embedded Mouse Tissue
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The segments of the tissues of 3 month-old mice were perfused and fixed with 4% paraformaldehyde in PBS under physiological pressure, and embedded in paraffin and sectioned at a thickness of 2–3 µm. TUNEL reactions were performed using TUNEL Enzyme, TUNEL Dilution Buffer and Biotin-16-dUTP (Roche Diagnostics Corp., Tokyo, Japan) according to the manufacturer's instructions. Sections were then incubated with anti-streptAvidin-HRP conjugated antibody (1:40 dilution), developed using diaminobenzidine [20 mg DAB, 65 mg NaN3, 17 µl H2O2 in 100 ml 0.05 M Tris–HCl buffer], and treated in methyl green nucleus staining.
6
Quantifying Cell Death in Organ Tissues
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Mice were sacrificed 12 h after the last application of anti-ceramide antibodies or phosphatidic acid. The heart, lung, liver, and kidney were removed, and frozen sections were obtained. Sections were air-dried for 5 min, fixed in ice-cold acetone for 5 min, and cell death was determined using TUNEL staining. To this end, sections were treated in 0.1 M sodium citrate (pH 6.0) in a microwave at 450 W for 5 min, washed twice in PBS, and incubated with each 5 μl TUNEL enzyme, 20 μl tetramethylrhodamine labeling, and 25 μl TUNEL dilution buffer (all from Roche) according to the vendor’s instructions (Roche). Samples were incubated for 60 min at 37 °C and washed three times in PBS. The samples were then incubated in PBS at 70 °C for 10 min to reduce background staining, washed once in PBS, and finally embedded in Mowiol (Sigma, Deisenhofen, Germany). Incubating the samples with DNAse served as a positive control for the TUNEL reaction. We then analyzed at least 500 cells in the tissues and determined the percentage of TUNEL-positive cells. The investigator was blinded to the identity of the samples. Samples were analyzed on a LEICA TCS SL microscope.
7
Quantification of BMEC Apoptosis by TUNEL
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The rate of BMEC apoptosis was evaluated using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining with an In Situ Cell Death Detection Kit (Roche, Basel, Switzerland). The cells were fixed with 4% paraformaldehyde for 30 min and then washed with PBS. Next, 0.3% Triton-X 100 in PBS was added and incubated for 5 min. The cells were rinsed 3 times with PBS. Then, the cells were incubated with 50 μL TUNEL Dilution Buffer (Roche-11966006001; Roche) in a 37°C humidified chamber for 1 h. The nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI) for 5 min at room temperature in the dark and later mounted with an anti-fade mounting medium. A fluorescent microscope (Olympus CX23; Olympus Corp., Tokyo, Japan) was used to capture images. Three independent investigators separately quantified the number of apoptotic cells based on the number of TUNEL-positive cells.
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