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% H₂O₂ 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 H₂O 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.

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).

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