Promega DeadEnd G7360 User manual

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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 [phone] or 800-356-9526 • Fax [phone]
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. WWaasshh::Immerse slides twice in PBS, 5 minutes each time.
3. PPeerrmmeeaabbiilliizzee::Immerse slides in 0.2% Triton®X-100 in PBS for 5 minutes.
4. WWaasshh::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. SSttooppRReeaaccttiioonn::Remove Plastic Coverslips. Immerse slides in 2X SSC for
15 minutes.
8. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
9. BBlloocckk::Immerse slides in 0.3% hydrogen peroxide for 3–5 minutes.
10. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
11. BBiinndd::Add 100µl Streptavidin HRP (diluted 1:500 in PBS). Incubate slides for
30 minutes at room temperature.
12. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
13. SSttaai
inn::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. WWaasshh::Immerse slides several times in deionized water.
15. VViissuuaalliizzee::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. PROTOCOL
Quick

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••
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••••
•••••
••••••
•••••••
© 2001–2009 Promega Corporation. All Rights Reserved. Printed in USA. Revised 3/09
Part #9FB056
ORDERING/TECHNICAL INFORMATION:
www.promega.com • Phone [phone] or 800-356-9526 • Fax [phone]
Apoptosis Detection in Tissue Sections
DeadEnd™ Colorimetric Apoptosis Detection System
INSTRUCTIONS FOR USE OF PRODUCTS G7130 AND G7360.
FOR LABORATORY USE. PROTOCOL
Quick
Prepare tissue-
containing slides.
Fix, permeabilize
and repeat fix.
Equilibrate and
label.
Stop reaction.
Block, bind
and stain.
Analyze with a
light microscope.
3219MA01_1A
Pretreatment of Tissue Sections
Perform Steps 1–4 for paraffin-embedded tissue sections. For frozen and
vibratome tissue sections, perform only Step 4.
1. RReemmoovveePPaarraaffffiinn::Wash slides twice in xylene, 5 minutes each time.
2. WWaasshh::Immerse in 100% ethanol for 5 minutes.
3. RReehhyyddrraattee::Wash slides in decreasing concentrations of ethanol (100%, 95%,
85%, 70%, 50%), 3 minutes each time.
4. WWaasshh::Immerse in 0.85% NaCl for 5 minutes. Immerse in PBS for 5 minutes.
Apoptosis Detection
1. FFiixx::Immerse slides in 4% paraformaldehyde in PBS for 15 minutes.
2. WWaasshh::Immerse slides twice in PBS, 5 minutes each time.
3. PPeerrmmeeaabbiilliizzee::Add 100µl of a 20µg/ml Proteinase K solution. Incubate at
room temperature for 10–30 minutes.
4. WWaasshh::Immerse slides in PBS for 5 minutes.
5. RReeppeeaatt
FFiixx::Immerse slides in 4% paraformaldehyde in PBS for 5 minutes.
6. WWaasshh::Immerse slides twice in PBS, 5 minutes each time.
7. EEqquuiilliibbrraattee::Add 100µl Equilibration Buffer. Equilibrate at room temperature
for 5–10 minutes.
8. LLaabbeell::Add 100µl of TdT reaction mix to the tissue sections on the slides.
Do not allow tissue sections 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.
9. SSttooppRReeaaccttiioonn::Remove Plastic Coverslips. Immerse slides in 2X SSC for
15 minutes.
10. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
11. BBlloocckk::Immerse slides in 0.3% hydrogen peroxide for 3–5 minutes.
12. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
13. BBiinndd::Add100µl Streptavidin HRP (diluted 1:500 in PBS). Incubate slides for
30 minutes at room temperature.
14. WWaasshh::Immerse slides three times in PBS, 5 minutes each time.
15. SSttaaiinn::Add 100µl DAB (prepared immediately prior to use). Develop until a
light brown background appears. Do not allow the background to become
too dark.
16. WWaasshh::Immerse slides several times in deionized water.
17. VViissuuaalliizzee::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