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Printed in USA. Revised 3/09.
Part #9FB056
© 2001–2009 Promega Corporation. All Rights Reserved.
ORDERING / TECHNICAL INFORMATION:
www.promega.com • Phone 608-274-4330 or 800-356-9526 • Fax 608-277-2601
Preparation of Slides
Grow cells on chamber slides or cytospin/pipette cells onto poly-
L
-lysine-coated
slides.
Apoptosis Detection
1. FFiixx:: Immerse slides in 10% buffered formalin or 4% paraformaldehyde for
25 minutes.
2. W
Waasshh:: Immerse slides twice in PBS, 5 minutes each time.
3. P
Peerrm
meeaabbiilliizzee:: Immerse slides in 0.2% Triton
®
X-100 in PBS for 5 minutes.
4. W
Waasshh:: Immerse slides twice in PBS, 5 minutes each time.
5. EEqquuiilliibbrraattee:: Add 100µl Equilibration Buffer. Equilibrate at room temperature
for 5–10 minutes.
6. LLaabbeell:: Add 100µl of TdT reaction mix to the cells on the slides. Do not allow
cells to dry completely. Cover slides with Plastic Coverslips to ensure even
distribution of the mix. Incubate slides for 60 minutes at 37°C in a humidified
chamber.
7. S
Sttoopp R
Reeaaccttiioonn:: Remove Plastic Coverslips. Immerse slides in 2X SSC for
15 minutes.
8. W
Waasshh:: Immerse slides three times in PBS, 5 minutes each time.
9. B
Blloocckk:: Immerse slides in 0.3% hydrogen peroxide for 3–5 minutes.
10. W
Waasshh:: Immerse slides three times in PBS, 5 minutes each time.
11. B
Biinndd:: Add 100µl Streptavidin HRP (diluted 1:500 in PBS). Incubate slides for
30 minutes at room temperature.
12. W
Waasshh:: Immerse slides three times in PBS, 5 minutes each time.
13. S
Sttaaiinn:: Add 100µl DAB (prepared immediately prior to use). Develop until a
light brown background appears. Do not allow the background to become
too dark.
14. W
Waasshh:: Immerse slides several times in deionized water.
15. V
Viissuuaalliizzee:: Mount slides in an aqueous or permanent mounting medium.
Observe staining with a light microscope.
See additional protocol information in Technical Bulletin #TB199, available
online at:
www.promega.com/tbs
Prepare cells
on slides.
Fix and
permeabilize cells.
Equilibrate and
label.
Stop reaction.
Block, bind
and stain.
Analyze with a
light microscope.
3218MA01_1A
Apoptosis Detection in Cultured Cells
DeadEnd™ Colorimetric Apoptosis Detection System
INSTRUCTIONS FOR USE OF PRODUCTS G7130 AND G7360.
FOR LABORATORY USE.
P R O T O C O L
Quick
