LSC Applicaton: Tissue Mapping of Apoptosis (TUNEL) in Murine Tissue

Solid tumor and tissue sections (4-6 µ) can be analyzed for DNA-fragmentation using TUNEL. The percentage of apoptotic nuclei can be quantitated and false-color imaged to create a bit-map for localization of apoptosis. Total nuclei are detected using a low concentration of propidium iodide stain and scanning in the Long Red. Apoptotic nuclei are detected in the Green. By assigning coloration according to Green fluorescence intensity, areas of high apoptosis can be easily visualized to help interpret drug effects within the context of tumor or tissue architecture. These areas can then be retrospectively relocated using CompuSort™ for image capture.

Apoptosis Analysis

Apoptosis analysis
with Apoptag staining

By use of tissue treatment negative controls and TUNEL-assay negative controls, a Green fluorescence negative gate is established (blue colored dots). TUNEL positive nuclei are colored as green or red dots depending on fluorescence intensity. Red color assigned nuclei correspond to intense staining observed with Apoptag™ immunohistochemical staining in serial tissue sections, whereas green color assigned nuclei correspond with weak Apoptag staining. Data show the effect and depth of penetration of an apoptosis-inducing compound administered i.p. to a human xenograft tumor in a SCID mouse.

Tissue Mapping

. Tumor map

.

Tissue Preparation and Staining
Snap-frozen tissue is sectioned (4-6 µ) using a cryostat microtome, and sections are fixed in 10% buffered formalin for 10 minutes. Slides can be stored at - 80° C before further staining. To stain for apoptosis, slides are first fixed again using cold ethanol/acetic acid (v:v 2:1) for 10 minutes at -20° C and then washed twice in PBS. TdT directed FITC-dUTP nick-end labeling is performed per kit instructions (Boehringer-Mannheim). After labeling, wash slides three times with PBS, 5 minutes for each wash. For nuclear staining, use a solution of propidium iodide made to 0.001% in PBS-EDTA/Triton X-100 containing 1:7 v:v dilution with Rnase A (Sigma). Incubate slides in the PI solution for 10 minutes in the dark. Wash slides twice in PBS. Mount slides with Vectashield™ mounting medium (Vector Labs).
LSC Analysis
The desired area of analysis is located visually using epi-fluorescence visual microscopy on the instrument. Scan areas are set and the scan initiated using the 20X objective and the argon laser operating at 5 mW. Individual nuclei are contoured using Long Red fluorescence and a 100 minimum pixel area threshold. Contouring outside of the nuclear area was kept to a minimum so that only nuclear events were detectable. Negative control tissue and TUNEL negative control reaction on a serial section of the test tissue were used to define the TUNEL negative gate for false-color assignments.
Data are from Dr. Michael Grace, Dr. Lei Xie, and Dr. Loretta Nielsen, Schering-Plough Research Inst., Kenilworth, N.J.