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TUNEL Apo-Green Detection Kit
Description
DNA fragmentation represents a characteristic of late stage apoptosis. DNA fragmentation in apoptotic cells can be detected by terminal
deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL).BiotoolTM TUNEL Apo-Green Detection Kit is a modified
FITC-12-dUTP TUNEL Assay designed to provide simple, accurate and rapid detection of apoptotic cells. It measures nuclear DNA
fragmentation, an important biochemical indicator of apoptosis by utilizing FITC-12-dUTP. FITC-12-dUTP is more readily incorporated into DNA
strand breaks than other larger ligands, giving rise to brighter signal that is sensitive and more accurate than traditional ligands. It also can be
used to assay apoptotic cell death in both tissue sections and cultured cells.
Storage
Components
Contents
Cat#:B31112
Cat#:B31115
Cat#:B31118
5x Equilibration Buffer
1.25 ml
1.25 ml×x 2
1.25 ml x 3
Apo-Green Labeling Mix
100 µl
250 µl
250 µl x 2
Recombinant TdT Enzyme
20 µl
50 µl
50 µl x×2
Proteinase K (2 mg/ml)
40 µl
100 µl
100 µl×x 2
1. Store the kit at -15°C to -25°C until the first use. After the first use,
if the kit will be used within one month, store the Recombinant TdT
Enzyme at -15°C to -25°C. Store the remaining components at 2°C to
8°C.
2. Protect the Apo-Green Labeling Mix in the Reaction Buffer from
unnecessary exposure to light.
Notice
Protocol
Please centrifuge before use！
Brief Summary
Cell suspensions
Adherent cells,
cell smears,
cytospins
Frozen tissue sections
Formalin-fixed,
paraffin-embedded
tissue sections
Dewax, rehydrate, and
treat with protease
Proteinase K
Fixation (1 h, 15-25°C)
Wash samples
Permeabilize samples in PBS containing 0.2%
Triton x-100 (5 min at room temperature)
Wash samples,then cover the samples with 1 x Equlibration Buffer (5-10min)
Incubate with TUNEL reaction mixture [enzyme solution + labeling solution]
(60
37°C)
Washmin,
samples
Wash samples
Stain the nucleus with DAPI or Hoechst (5 min, room temperature)
Analyze by flow
cytometry or
fluorescence
microscopy
Analyze by fluorescence microscopy or
laser scanning confocal microscopy
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]
Preparation of Cell Suspensions, adherent cells and Frozen
tissue sections
1. Fix samples by immersing slides in freshly prepared 4%
paraformaldehyde solution in PBS (pH 7.4) in a Coplin jar for 25
minutes at 4°C.
2. Wash the samples by immersing in fresh PBS for 5 minutes.
Repeat PBS wash twice.
3. Permeabilize samples by immersing the slides in 0.2% Triton X-100
solution in PBS for 5 minutes at room temperature.
4. Wash the samples in PBS for 5 minutes.
5. Remove excess liquid then cover the samples with 100µl of
1 x× Equilibration Buffer at room temperature for 5-10 minutes.
6. Prepare reaction mixture before use based on the number of
samples to be assayed.(see table below)
Reaction Components
50 µL Reaction Volume (µL)
ddH2O
34
5 x×Equilibration Buffer
10
Apo-Green Labeling Mix
5
Recombinant TdT Enzyme
1
7. Add 50 µL of the reaction mixture to each sample and incubate at
37°C for 60 minutes(Avoid exposing the samples to light).
Remove the reaction mixture, and wash the sample 3 times with
PBS.
8. Stain the nucleus with DAPI or Hoechst for image analysis.
Wash the samples in PBS for 5 minutes at room temperature.
9. Alternatively, add one drop of Anti-fluorescence quencher to the
10. sample area and mount slides using glass coverslips.
11. Immediately analyze samples under a fluorescence microscope or
laser scanning confocal microscope using a standard fluorescein
12. filter set to view the Apo-green fluorescence at 520 ± 20nm and
view blue DAPI at 460nm.
Pretreatment of Paraffin-Embedded and Formalin-fixed Tissue
Sections
1. Dewax tissue sections by immersing slides in fresh xylene in a
Coplin jar for 5 minutes at room temperature.Repeat one time for
a total of two xylene washes.
2. Wash the samples by immersing the slides in 100% ethanol for 5
minutes at room temperature in a Coplin jar.
3. Rehydrate the samples by sequentially immersing the slides
through graded ethanol washes (100%, 95%, 85%, 70%, 50%) for
5 minutes each at room temperature.
4. Wash the samples 3 times in PBS for 5 minutes at room
temperature.
5. Remove the liquid then place the slides on a flat surface. Prepare
a 20 µg/ml Proteinase K solution from the Proteinase K(2 mg/ml)
by diluting 1:100 in PBS. Add 100 µl of the 20 µg/ml Proteinase K
to each slide to cover the tissue section. Incubate slides for 8–20
minutes at room temperature.
6. Note: Proteinase K helps permeabilize tissues and cells to the
staining reagents in subsequent steps. For best results, optimize
the length of incubati on with Proteinase K. Longer incubations
may be needed for tissue sections thicker than 4–6 µm; however,
with prolonged Proteinase K incubations, the risk of releasing the
tissue sections from the slides increases in subsequent wash
steps.
7. Wash the samples by immersing the slides in PBS for 5 minutes
at room temperature.
8. 8Incubate samples with 1 x Equilibration Buffer at room
temperature for 5-10 minutes.
9. Prepare reaction mixture before use based on the number of
samples to be assayed.(see table below)
Reaction Components
50 µL Reaction Volume (µL)
ddH2O
34
5 × Equilibration Buffer
10
Apo-Green Labeling Mix
5
Recombinant TdT Enzyme
1
10. Add 50 µL of the reaction mixture to each sample and incubate at
37°C for 60 minutes(Avoid exposing the samples to light).
11. Remove the reaction mixture, and wash the sample 3 times with
PBS.
12. Stain the nucleus with DAPI or Hoechst for image analysis.
13. Wash the samples in PBS for 5 minutes at room temperature.
14. Alternatively, add one drop of Anti-fluorescence quencher to the
sample area and mount slides using glass coverslips.
15. Immediately analyze samples under a fluorescence microscope
or laser scanning confocal microscope using a standard
fluorescein filter set to view the Apo-green fluorescence at 520 ±
20 nm and view blue DAPI at 460 nm.
Troubleshooting
Please review the following for trouble-shooting options when you
encounter technical difficulties.
Problem
Potential Cause (s)
Suggestion (s)
High
background
Nonspecific incorporation of FITC-12dUTP. Do not allow the cells or slides
to dry out.
Slides may be washed three
times for 5 minutes with PBS
containing 0.1%Triton X-100.
Little or poor
labeling
Insufficient permeabilization with
Triton X-100 or Proteinase K.
Optimize permeabilization step
by adjusting incubation time
with permeabilization agent.
Loss of tissue
section from
the slides
Insufficient coating of the slides prior to
attachment of tissue section. Coat
microscopic slides with poly-L-lysine
before spreading the tissue sections.
To prevent tissue section being
enzymatically digested from the
slide, optimize the Proteinase K
incubation time.
Few cells
remaining for
High number of cells lost during the
the final
microscopic or procedure.
flow cytometric
analysis
Start with a higher number of
cells.
When preparing a cell
suspension for attachment to
microscope slides, please
wash cells with PBS containing
1% BSA during centrifugation.
Use a cytospin centrifuge to
attach the cells to microscope
available.
When preparing cells in
suspension, please wash cells
with PBS containing 1% BSA
during centrifugation.
Order & Inquiry
Order & Inquiry
Tel: (713)732-2181
Fax: +1-866-747-4781
E-mail: [email protected]
Tel: +49-89-46148500
Fax: +49-89-461485022
E-mail: [email protected]