ab66110
In situ BrdU-Red DNA
Fragmentation (TUNEL)
Assay Kit
Instructions for Use
For the rapid, sensitive and accurate
measurement of apoptosis in various samples
This product is for research use only and is not
intended for diagnostic use.
Table of Contents
Table of Contents
2
1.
Overview
3
2.
Protocol Summary
4
3.
Components and Storage
6
4.
Assay Protocol for IHC-P
9
1. Overview
Internucleosomal DNA fragmentation is a hallmark of apoptosis in
mammalian cells. Abcam’s Red Fluorescence In situ BrdU DNA
Fragmentation (TUNEL) Assay Kit provides complete components
including positive and negative control cells for conveniently
detecting DNA fragmentation by fluorescence microscopy.
The kit utilizes Br-dUTP (bromolated deoxyuridine triphosphate
nucleotides) which is more readily incorporated into DNA strand
breaks than other larger ligands (e.g., fluorescein, biotin or
digoxigenin). The greater incorporation gives rise to brighter signal
when the Br-dUTP sites are identified by a Red fluorescence labeled
anti-BrdU monoclonal antibody. The assay is suitable for studying
apoptosis with GFP transfected cells.
2. Protocol Summary
IHC-P DETECTION
Prepare Tissue Sections in Coplin Jars
Fix with 4% Formaldehyde (fresh frozen tissues only)
Wash Cells with PBS
Add Proteinase K solution
Wash Cells with PBS
Wash Cells with Wash Buffer
Prepare and Add DNA Labeling Solution
Rinse Cells
Add Antibody Solution
Add 7-AAD/ RNase A Solution
Rinse Cells
Seal coverslip and slides, view results with Fluorescent Microscope
3. Components and Storage
A. Kit Components
Item
Quantity
Storage
Temp.
Positive Control Cells
5 mL
-20°C
Negative Control Cells
5 mL
-20°C
Wash Buffer
120 mL
+4°C
Reaction Buffer
0.6 mL
+4°C
45 μL
-20°C
Br-dUTP
0.48 mL
-20°C
Rinse Buffer
120 mL
+4°C
Anti-Brdu (Red) Antibody
0.3 mL
+4°C
7-AAD/RNase Staining Buffer
30 mL
+4°C
TdT Enzyme
* Store components separately according Table A.
B. Additional Materials Required

Double-distilled water (ddH2O)

Microcentrifuge

PBS

Pipettes and pipette tips

4% Formaldehyde

Orbital Shaker

70% Ethanol
FOR IHC-P

Coplin jars

Xylene

Ethanol in the following percentages: 100% - 95% - 85% 70% - 50%

Coverslips (plastic and glass)

0.85% NaCl solution

10 mg/ml Proteinase K

100 mM Tris-HCl, pH 8.0 + 50mM EDTA solution

(Optional) Anti-fading mounting solution (we recommend
Fluoroshield Mounting Medium (ab104136))

(Optional)Nail
polish
or
rubber
cement
4. Assay Protocol for IHC-P
IHC-P is not a tested application for this product and this
protocol might require further optimization.
1. Tissue Section Preparations:
Note: This protocol describes the preparation of formalin-fixed,
paraffin-embedded apoptotic tissue section mounted on glass slides.
If using fresh-frozen tissue sections, proceed directly to Step G.
a) Remove paraffin by immersing slides in a Coplin jar containing
fresh xylene. Incubate 5 min at RT.
b) Repeat previous step in a second Coplin jar containing fresh
xylene.
c) Immerse slides in a Coplin jar containing 100% ethanol and
incubate 5 min at RT.
d) Rehydrate slides by sequential 3-min, RT incubations in Coplin
jars containing:

100% ethanol

95% ethanol

85% ethanol

70% ethanol

50% ethanol
e) Immerse slides in a Coplin jar containing 0.85% NaCl and
incubate 5min at RT.
f) Immerse slides in a Coplin jar containing PBS and incubate 5 min
at RT.
g) For fresh-frozen tissue sections ONLY: Fix slides by
immersing
them
in
a
Coplin
jar
containing
fresh
4%
formaldehyde/PBS and incubate 15 min at RT.
h) Wash slides by immersing them in a Coplin jar containing PBS
and incubate 5 min at RT.
i) Repeat washing step in a new Coplin jar containing fresh PBS.
Allow liquid to drain thoroughly and place slides on a flat surface.
j) Prepare 20 µg/ml Proteinase K solution (2 µl Prot K 10 mg/ml +
998 µl 100 mM TrisHCl pH8.0 + 50 mM EDTA) and cover each
tissue section with 100 µl. Incubate 5 min at RT.
k) Immerse slides in a Coplin jar containing PBS and incubate 5 min
at RT.
l) Transfer slides to a Coplin jar containing 4% formaldehyde/PBS
and incubate 5 min at RT.
m) Immerse slides in a Coplin jar containing PBS and incubate 5 min
at RT.
2. Detection by Fluorescence Microscopy:
a) Remove slides from PBS and tap gently to remove excess liquid.
Cover section with 100 µl of Wash Buffer (blue cap).
b) Using forceps, gently place a piece of plastic coverslip on top of
the cells to evenly spread the liquid and incubate 5 min at RT.
Remove plastic coverslip and gently tap the slides to remove
excess liquid.
c) Repeat previous step. Carefully blot dry around the edges with
tissue paper.
d) Cover slides with 50 μl of the DNA Labeling Solution prepared as
described below:
DNA Labeling Solution
TdT Reaction Buffer
TdT Enzyme
Br-dUTP
ddH2O
Total Volume
1 assay
10 assays
10 μL
100 μL
0.75 μL
7.5 μL
8 μL
80 μL
32.25 μL
322.5 μL
51 μL
510 μL
e) Using forceps, gently place a piece of plastic coverslip on top of
the cells to evenly spread the liquid. Place slides in a dark,
humidified 37°C incubator for 1 hour.
Note: ensure high humidity by placing wet paper towels in the
bottom of the dry incubator.
f) Remove the plastic coverslips using forceps. Rinse slides in a
fresh Coplin jar filled with PBS for 5 min.
g) Repeat previous washing step and carefully blot dry around the
edges with tissue paper.
h) Cover slides with 100 µl of the Antibody Solution prepared as
described below:
Antibody Solution
Anti-BrdU-Red Antibody
Rinse Buffer
1 assay
10 assays
5 μL
50 μL
95 μL
950 μL
i) Using forceps, gently place a piece of plastic coverslip on top of
the cells to evenly spread the liquid. Incubate slides in a dark,
humidified incubator for 30 min at RT.
j) Carefully remove plastic coverslips and solution for slides.
k) Add 100 µl of 7-AAD/RNase A Staining Buffer.
l) Using forceps, gently place a piece of plastic coverslip on top of
the cells to evenly spread the liquid. Incubate slides in a dark,
humidified incubator for 30 min at RT.
m) Wash cells by transferring slides to a fresh Coplin jar filled with
ddH2O and incubate for 5 min at RT.
n) Repeat previous washing step.
o) [Optional] Add a drop of anti-fading solution and cover the treated
portion of the slide with a glass coverslip.
p) [Optional] Seal the edges of the coverslip with rubber cement or
clear nail polish.
q) View slides as soon as possible.
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All information
/ detail
is correct
at time of going
to print.
Version
4 Last
Updated
9 December
2014