TUNEL and Active Caspase-3 Detection by IHC/ICC Protocol
This protocol is intended as a guide only.
This protocol is modified from the following kit: Steps 2-16 pertinent reagents are from Apo-BrdU-IHC Kit (catalog # NBP2-31164).
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
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
| Complete Labeling Reaction Mixture | 1 Sample |
|---|---|
| 5x Reaction Buffer | 10 µL |
| TdT Enzyme | 0.75 µL |
| Br-dUTP | 8 µL |
| diH2O | 32.25 µL |
| Total volume | 51 µL |
| Antibody Solution | 1 Sample |
|---|---|
| Biotin~PRB-1 | 5 µL |
| Blocking Buffer | 95 µL |
| Total volume | 100 µL |
| 1x Conjugation Solution | 1 Sample |
|---|---|
| 200x Conjugate | 0.5 µL |
| Blocking Buffer | 100 µL |
| Total volume | 100.5 µL |
Methods
- Induce apoptosis by desired method and include vehicle-treated cells/animal (negative control).
- 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.
Cover the entire specimen with 100 µL Proteinase K solution and incubate in a humidity chamber at RT at noted below. Do not over incubate
Paraffin-embedded tissue section 20 minutes Frozen tissue section 10 minutes Cells 5 minutes - Rinse slide with 1X PBS for 5 minutes.
- Block endogenous peroxidase by incubating specimen with 100 μl of 3% H2O2 for 10 minutes at RT.
- Rinse slide with 1X PBS for 5 minutes.
- 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.
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).
- Remove parafilm and rinse slide with 1X PBS for 5 minutes.
- Cover specimen with 100 µL with Blocking Buffer for 10 minutes at RT. Then carefully aspirate or blot solution.
- 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.
- Rinse slide with 1X PBS for 5 minutes.
- Cover specimen with 100 µL with Blocking Buffer for 10 minutes at RT. Then carefully aspirate or blot solution.
- Immediately cover specimen with 100 µL of 1X Conjugate Solution and incubate for 30 minutes at RT.
- Rinse slide with 1X PBS for 5 minutes.
- Incubate tissue section with DAB solution for up to 15 minutes at RT. Monitor nuclei staining (dark brown color) under the microscope.
- Wash slide in 1X PBS for 20 minutes.
- Block endogenous peroxidase by incubating specimen with 100 μL of 3% H2O2 for 10 minutes at RT.
- 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.
- Wash slide in 1X PBS for 5 minutes.
- Cover specimen with active caspase-3 Antibody Solution (5-15 μg/mL) and incubate overnight at 2-8 °C.
- Wash slide 3 times in 1X PBS for 15 minutes.
- Cover specimen with anti-rabbit secondary Antibody Solution for 30-60 minutes at RT.
- Wash slide 3 times in 1X PBS for 15 minutes.
- Incubate tissue section with HSS-HRP for 30 minutes at RT.
- Wash slide 3 times in 1X PBS for 2 minutes.
- Incubate tissue section for 2-5 minutes with AEC Chromogen. Monitor red color staining development under the microscope.
- Rinse slide with diH2O and counterstain with Methyl Green.
- Rinse slide with PBS, mount using Aqueous Mounting Medium, and then dry the slide.
- Image with a bright field microscope.