TUNEL assay for apoptosis:

Apoptosis is associated with physiological or programmed cell death, in contrast with necrosis which is associated with cell injury. Some examples of systems in which apoptosis occurs are:Maturation of the immune system, embryonic development, hormone deprivation of endocrine or other hormone dependent or sensitive cells, and normal tissue turnover.

Residues of FITC labeled dUTP, or biotin dUTP are catalytically added to the DNA by TdT, an enzyme which catalyses a template independent addition of dUTP to the 3-OH ends of double or single stranded DNA. The biotinylated nucleotides are then detected by using labeled Streptavidin. The fluorochrome CY3 is similar to rhodamine but is brighter and appears to be more stable to room temperature storage for upto 3 months. The enzyme label HRP (Horse Radish Peroxidase) may also be used for detection, and needs visualization by addition of appropriate substrates. The apoptotic bodies within nuclei of the tissue sections, are clearly visible above background extremely well.

REMEMBER TO USE Sections of normal SPLEEN or GUT or SKIN as POSITIVE Controls for all TUNEL assays, to make sure the assay produces expected results.

Reagents:

1. On PARAFFIN sections we use the TUNEL Kit from BD Biosciences: Cat. No. K2024-2 .

   If using this kit, the paraffin sections need to be treated with Proteinase K for 5 minutes, rinsed with buffer, before proceeding, following manufacturer's instructions.

   Another good kit is from Roche Catalog # 1-684-795, Clontech Catalog #K2024-2

Clontech cat. # 6301083 can be used for both frozen and paraffin sections.

2. On FROZEN sections, we use Promega Cat.#M1871 (contains 5xTdT buffer, TdT enzyme, 5M NaCl)
3. Biotin dUTP: Boehringer Manheim Cat #1093070
4. CY3 Streptavidin (Jackson labs Cat#016-160-084)
5. Acetone: Fisher Cat#A16P-4
6. Phosphate buffered saline pH 7.2 (PBS)
7. 1% BSA (bovine serum albumin) in PBS: BSA Fisher Cat# A2153
8. Vectashield coverslipping medium (Vector Cat# H1200)

Procedure:

1. Five micron thick frozen sections are cut and airdried at 4oC for 48 hours. Following a cold acetone fixation for 10 minutes, the slides are airdried for 2 minutes before 3 rinses in PBS.
2. The slides are then rinsed in 1x TdT buffer for 5 minutes before careful application of the TdT reation mix to the tissue sections on each slide, which are then incubated in a humid chamber at 37oC for 60 minutes, followed by 3 PBS washes.
3. The CY3-Streptavidin is diluted in 1%BSA/PBs and applied to the sections, which are then incubated for 30 minutes at room temperature, followed by 3 PBS washes.
4. The slides are then coverslipped using Vectashield for viewing on the Zeiss epifluorescent microscope.