In Situ Cell Death (Apoptosis) Detection by TUNEL labeling

Protocol for Paraffin Sections:

Dewax paraffin sections:

Incubate slides, 55°C, 30 min.
      Xylenes, 2 times, 2 min. each
      100% EtOH, 2 times, 2 min. each
      95% EtOH, 2 times, 2 min. each
      80% EtOH, 2 min.
      75% EtOH, 2 min.
      50% EtOH, 2 min.
      dH₂O rinse.

Incubate slides in a 1µg/ml Proteinase K/10mM Tris solution, 15 min., RT. (7.5µl of 20µg/µl PK in 150 ml 10mM Tris, pH 7.4-8.0).

All slides: 1x PBS rinse, 2 times (+ 10 min for those non-positive control slides).

(Positive control slide: in DNase I solution (100µl of 200µg/ml), 10 min., RT. 1x PBS rinse, 2 times in a separate container then combine with other slides.)

Wipe around tissue.

Make up negative Control solution (just Label solution containing FITC) and TUNEL solutions at time of use:

A. Remove 100µl from Tube 2 (Label solution) for 2 negative controls (50µl each). Do this even if you are omitting this negative control so that volumes and concentrations will remain consistent for the labeling.

B. Add Total volume (50µl,) of Tube 1 (TdT) + remainder of Tube 2 (450µl).
      Apply 100µl TUNEL reaction mixture (or 100µl Control Label solution for negative control) to each slide. 
      Incubate in humid chamber, 60 min., 37°C. 
      1x PBS wash, 3 times. 
      Wipe around tissue. 
      Apply 100µl anti-FITC-AP conj. ("converter-AP") on each sample. 
      Incubate in humid chamber, 30 min., 37°C. 
      1x PBS wash, 3 times. 
      100mM Tris buffer, pH 8.2, 5 min., RT. 
      Add 50-100µl substrate solution (5-6 drops Vector Blue or Vector Red substrate/per slide):

Mix: 
      5ml 100mM Tris, pH 8.2
      1 drop Levamisole
      2 drops each of Solution 1, 2, and 3 of either Vector substrate
      Incubate in absence of light, RT. Vector Blue - 10 min.; Vector Red - 5-8 min. 
      dH₂O, 1 time to stop color reaction. 

Counterstain for Vector Blue: 
      Gill's Hematoxylin, No. 2, 5 sec.
      Water rinse until clear
      Scott's solution, 20 sec.
      Water rinse until clear
      70% EtOH, 30 sec.
      95% EtOH, 2 times, 30 sec. each
      100% EtOH, 2 times, 30 sec. each
      Histoclear, 1 min.
      Histoclear, 1 min.
      Coverslip with Accumount medium.

Counterstain for Vector Red: 
      Gill's Hematoxylin, No. 2, 5 sec.
      Water rinse until clear
      Scott's solution, 20 sec.
      Water rinse until clear
      70% EtOH, 30 sec.
      95% EtOH, 2 times, 30 sec. each
      100% EtOH, 2 times, 30 sec. each
      Xylenes, 1 min.
      Xylenes, 1 min.
      Coverslip with Accumount medium.

Reagents Needed: 
      3L 1x PBS
      10mM Tris-HCl
      20 mg/ml Proteinase K
      100µl/slide x-phosphate/BCIP or Fast Red substrate
      DNase I solution (1mg/ml - 1µg/ml) for Positive control
      100mM Tris-Hcl, pH 8.2