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This protocol is modified from the following kits: Steps 2-16 pertinent reagents are from Apo-BrdU-IHC Kit (catalog # NBP2-31164). Steps 17-29 (except Caspase 3 antibody) and pertinent reagents are from anti-rabbit HRP-AEC IHC Detection Kit (catalog # CTS006-NOV).
MATERIALS
NOT supplied in kits:
> 1X PBS
> 1X PBS, 1mM MgSO4
> 10mM Tris pH 8.0
> DNase I
> 3% H2O2 – in methanol
> anti-active caspase-3 antibody (catalog # AF835)
> Aqueous Mounting Medium (catalog # CTS011-NOV)
Apo-BrdU-IHC Kit:
> Proteinase K – in 10mM Tris pH 8.0 (100x dilution before use)
> 1X Reaction Buffer (10x dilution before use)
> Blocking Buffer
> DAB in H2O2/Urea (prepare immediately before use)
> Methyl Green
HRP-AEC detection kit:
> Avidin Blocking Reagent
> Biotin Blocking Reagent
> Biotinylated Secondary Antibody
> HSS-HRP
> AEC Chromogen and Buffer (prepare immediately before use)
Optional: Preparation of positive TUNEL control - Nuclease treated sample
> Cover the entire specimen with 1 µg/ml DNase I in 1X PBS, 1mM MgSO4 and incubate at room temperature (RT) for 20 min
> Rinse with 1X PBS
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Complete Labeling Reaction Mixture
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1 Sample
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5x Reaction Buffer
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10 µl
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TdT Enzyme
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0.75 µl
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Br-dUTP
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8 µl
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diH2O
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32.25 µl
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Total volume
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51 µl
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Antibody Solution
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1 Sample
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Biotin~PRB-1
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5 µl
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Blocking Buffer
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95 µl
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Total volume
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100 µl
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1x Conjugation Solution
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1 Sample
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200x Conjugate
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0.5 µl
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Blocking Buffer
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100 µl
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Total volume
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100.5 µl
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METHODS
1. Induce apoptosis by desired method and include vehicle-treated cells/animal (negative control).
2. Following standard procedures to prepare cells or tissue sections for ICC/IHC. Wash the specimen in 1X PBS. Do not let cells/tissue sections dry out during or between any step! For help on preparing IHC samples, see our IHC handbook.
3. Cover the entire specimen with 100 µl Proteinase K solution and incubate in a humidity chamber at RT at noted below. Do not overincubate.
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Paraffin-embedded tissue section
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20 minutes
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Frozen tissue section
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10 minutes
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Cells
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5 minutes
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4. Rinse slide with 1X PBS for 5 minutes.
5. Block endogenous peroxidase by incubating specimen with 100 μl of 3% H2O2 for 10 minutes at RT.
6. Rinse slide with 1X PBS for 5 minutes.
7. Cover specimen with 100 µl 1X Reaction Buffer and incubate for 10-30 minutes at RT. Then carefully aspirate or blot solution. Prepare Complete Labeling Reaction Mixture during the incubation.
8. Immediately apply 50 µl of Complete Labeling Reaction and cover the specimen with a piece of parafilm, cut slightly larger than the specimen. Incubate in a humidity chamber for 1 to 1.5 hours at 37°C. The parafilm prevents evaporation and folding up one corner aids in its application/removal.
Note: Depending on the tissue and fixation conditions, the incubation period of the DNA End Labeling Reaction may need to be adjusted (shortened or lengthened).
9. Remove parafilm and rinse slide with 1X PBS for 5 minutes.
10. Cover specimen with 100 µl with Blocking Buffer for 10 minutes at RT. Then carefully aspirate or blot solution.
11. Immediately apply 100 µl of Antibody Solution onto the specimen and incubate for 1 to 1.5 hours in the dark at RT. Cover slides with aluminum foil.
12. Rinse slide with 1X PBS for 5 minutes.
13. Cover specimen with 100 µl with Blocking Buffer for 10 minutes at RT. Then carefully aspirate or blot solution.
14. Immediately cover specimen with 100 µl of 1X Conjugate Solution and incubate for 30 minutes at RT.
15. Rinse slide with 1X PBS for 5 minutes.
16. Incubate tissue section with DAB solution for up to 15 minutes at RT. Monitor nuclei staining (dark brown color) under the microscope.
17. Wash slide in 1X PBS for 20 minutes.
18. Block endogenous peroxidase by incubating specimen with 100ul of 3% H2O2 for 10 minutes at RT.
19. Rinse slide in 1X PBS and block endogenous biotin. Incubate slide with Avidin Blocking Reagent for 15 minutes, followed by a 15 minute incubation with Biotin Blocking Reagent.
20. Wash slide in 1X PBS for 5 minutes.
21. Cover specimen with active caspase-3 Antibody Solution (5-15 μg/ml) and incubate overnight at 2-8 °C.
22. Wash slide 3 times in 1X PBS for 15 minutes.
23. Cover specimen with anti-rabbit secondary Antibody Solution for 30-60 minutes at RT.
24. Wash slide 3 times in 1X PBS for 15 minutes.
25. Incubate tissue section with HSS-HRP for 30 minutes at RT.
26. Wash slide 3 times in 1X PBS for 2 minutes.
27. Incubate tissue section for 2-5 minutes with AEC Chromogen. Monitor red color staining development under the microscope.
28. Rinse slide with diH2O and counterstain with Methyl Green.
29. Rinse slide with PBS, mount using Aqueous Mounting Medium, and then dry the slide.
30. Image with a bright field microscope.
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