Detection of Apoptosis in Paraffin-embedded tissues by ISEL (In Situ End Labeling)

Detection of Apoptosis in Paraffin-embedded tissues by  ISEL (In Situ End Labeling)

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.

Notes1)  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).