Detection of Apoptosis in Paraffin-embedded tissues by ISEL (In Situ End Labeling)
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<center> <font><font><font color="#000000"><b>Detection of Apoptosis in Paraffin-embedded tissues</b><br /> <b>by ISEL (In Situ End Labeling)</b> </font> </font> </font></center>
The ISEL technique identifies morphologically apparent karyorrhetic cells and also labels cells in which the DNA fragmentation has not yet progressed to frank nuclear fragmentation. By using biotinylated nucleotides and a DNA polymerase, the DNA fragments serve as templates in situ to synthesize new DNA strands, which are visualized by light microscopy after histochemical processing.
Reference: Wijsman et al., J of Histochem. Cytochem. 41(1):7-12, 1993.
PROCEDURE
To determine the optimal conditions for each study, perform the following “titration” using the standard protocol below. Serial sections from the same block provide the best samples for the titration.
SAMPLE dUTP dilution POL I dilution DIGESTION
1A 1:100 1:200 NONE
2A 1:250 1:200 NONE
3A 1:500 1:200 NONE
4A 1:100 1:500 NONE
5A 1:250 1:500 NONE
6A 1:500 1:500 NONE
1B 1:100 1:200 TRYPSIN
2B 1:250 1:200 TRYPSIN
3B 1:500 1:200 TRYPSIN
4B 1:100 1:500 TRYPSIN
5B 1:250 1:500 TRYPSIN
6B 1:500 1:500 TRYPSIN
1C 1:100 1:200 PEPSIN
2C 1:250 1:200 PEPSIN
3C 1:500 1:200 PEPSIN
4C 1:100 1:500 PEPSIN
5C 1:250 1:500 PEPSIN
6C 1:500 1:500 PEPSIN
A. DEPARAFFINIZE AND HYDRATE
5 min each in:
xylene X 2
xylene:EtOH (1:1)
100% EtOH
90% EtOH
70% EtOH
di H2O X 2
B. PRETREAT
Block endogenous peroxidase by immersing slides in 3% H2 O2 for 30 min at RT.
Prepare 2XSSC and preheat to 80°C.
Rinse slides in diH2O.
Wash in 0.15 M PBS 3 X 4 min
Incubate in the preheated 2 X SSC at 80°C for 20 min.
Rinse once in 0.15 M PBS, then wash in 0.15 M PBS 3 X 4 min.
If necessary as determined by titration, incubate slides under desired enzyme digestion conditions. Rinse once in 0.15 M PBS, then wash in 0.15 M PBS 3 X 4 min.
Equilibrate in Buffer A 3 X 4 min.
C. LABEL
Prepare ISEL solution , adding enzyme immediately before use.
Incubate slides in ISEL solution in 18°C ice bath for 2 hrs.
Prepare ABC solution near end of ISEL incubation, for use 30 to 60 minutes later.
Rinse in Buffer A, then wash in Buffer A 3 X 4 min.
Wash in 0.5 M PBS 3 X 4 min.
D. DETECT
Apply ABC solution and incubate at RT for 30 min.
Wash in 0.5 M PBS 3 X 10 min.
Prepare DAB+Ni .
Apply DAB for 3 to 10 min at RT.
Check reduction of DAB under microscope.
When color has developed to the desired intensity, rinse in running di H2 O for 2 minutes.
E. COUNTERSTAIN
Immerse in Hematoxylin for 90 sec.
Rinse in water.
Blue in Lithium chloride for 20 sec.
Rinse in water.
Rehydrate and coverslip.
Notes
1) In addition to the hematoxylin, a light eosin counterstain may be used to further aid in detection of apoptotic bodies using morphological criteria. It is best to use the DAB+Ni chromagen if using an H&E counterstain.
2) This procedure can be coupled with detection of BrdU. Follow the ISNT procedure up to the counterstaining step (section E). Then equilibrate slides in auto buffer and follow the BrdU staining procedure starting at the 2N HCl incubation. Use an AEC chromagen for the BrdU instead of DAB. A section of doudenum is an excellent positive control for this dual stain. This tissue should be stained black at the tips of the villi (ISEL) and red in the crypts (BrdU).
