Detection of apoptotic process in situ using immunocytochemical-2
B.2.1 Materials
proteinase K (pK) (A2), H2O2 , TdT buffer (A1), TdT enzyme (A2), biotinylated dUTP (A2), TB buffer (A1), serum albumin (BSA) (A2), PBS, Extra-avidin Peroxidase (A2), AEC solution (A1), DNAse buffer (A1).
- B.2.2. Methodology
1. Tissue sections are dewaxed and rehydrate by heating at 60°C.
2. Wash in xylene and rehydrate through a graded series of ethanol and deionized water.
3. Stripp from proteins by incubation with 20
mg/mL pK for 15 min at room temperature.
4. Wash in deionized water (4x2 min).
5. Cover sections with 2% H
2O
2 for 5 min at room temperature, to inactivate endogenous peroxidase.
6. Wash in deionized water.
7. Immerse in TdT buffer.
8. Add TdT (0.3 e.u./
mL) and biotinylated dUTP in TdT buffer to cover the sections.
9. Incubate in humid atmosphere at 37°C for 60 min.
10. Stop the reaction by transferring the slides to TB buffer for 15 min at room temperature.
11. Wash in deionized water.
12. Cover the sections with 2% aqueous solution of BSA for 10 min at room temperature.
13. Wash in deionized water.
14. Immerse in PBS for 5 min.
15. Cover the sections with Extra-avidin Peroxidase diluited 1:10-1:20 in water.
16. Incubate for 30 min at 37°C.
17. Wash in deionized water.
18. Stain with AEC solution for about 30 min at 37°C.
In this method, cytospin slides from cell suspension and cryopreserved tissues can also be utilized. For
cytospin slides: i) fix air dried cell samples with a freshly prepared paraformaldeyde solution (4% in PBS, pH 7.4) for 30 min at room temperature; ii) rinse slides with PBS and incubate in permeabilisation solution (0.1% Triton X-100, 0.1% sodium citrate) for 2 min in ice and go directly to step 4.
For cryopreserved tissues: i) fix tissue sections with 4% paraformaldeyde for 20 min at room temperature; ii) wash 30 min with PBS; iii) incubate slides in permeabilisation solution for 2 min in ice and go directly to step 4.
To perform each experiment using a blank, a negative and a positive control samples, is recommended. The blank sample is assessed passing from step 14 directly to step 17, in order to avoid the Extra-avidin Peroxidase incubation. The negative sample is assessed substituting step 8 as following: add biotinylated dUTP in TdT buffer to cover the sections (without TdT enzyme). The positive sample, in order to make sure that the method works, is assessed by digesting, after step 6, with DNAse buffer for 10 min at room temperature and wash extensively with deionized water before step 7.
The protocol require 3-4 hours excluding culture and preparation of tissue sections. Obviously, utilizing commercial kits the duration of method is reduced (2-3 hours for immunoenzymatic kit and 1-2 hours for fluorescein kit).
1. Gavrieli, Y., Sherman, Y., Ben-Sasson, S.A. 1992. Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation.
J. Cell Biol. 119: 493.
B.3. COMMENTARY
B.3.1 Background information
Moreover, as this procedure is quite complex, the use of commercial kits such as ApoTagTM(Oncor, Gaithersburg, MD, USA) and "In situ cell death detection Kit" (Boeringer-Mannheim, Germany), is recommended. Utilizing the last one, the incorporation of fluorescein-labeled nucleotides to DNA strands breaks, allows, using a fluorescence microscopy or a flow cytometer, to have a simple and fast method.
B.3.3 Time considerations
B.3.4 Key references
2. Gorczyca, W., Gong, J., Darzynkiewicz, Z. 1993. Detection of DNA strand breaks in individual apoptotic cells by the in situ terminal deoxynucleotidyl transferase and nick translation assays. Cancer Res. 52: 1945.
3. Gorczyca, W.,Tuziak,T., Kram, A., Melamed, M.R., Darzynkiewicz, Z. 1994. Detection of apoptosis-associated DNA strand breaks in fine-needle aspiration biopsies by in situ end labeling of fragmented DNA. Cytometry 15: 169.
4. Li, X., Darzynkiewicz, Z. 1995. Labelling DNA strand breaks with BrdUTP. Detection of apoptosis and cell proliferation. Cell Prolif. 28: 571.
| Solution | Preparation | Storage |
| A. Bouin's fixative | satured aqueous picric acid 37.5 mL; formalin (37-40%) 12.0 mL; glacial acetic acid 2.5 mL | utilize freshly prepared |
| B. TdT buffer | 30 mM Trizma base (pH 7.2), 140 mM sodium cacodylate, 1 mM cobalt chloride | 4°C |
| B. TB buffer | 300 mM sodium chloride, 30 mM sodium citrate | RT |
| B. AEC solution | 5 mg of AEC, 100 mL of N-N’-dimethylformamyde and add acetate buffer up to 10 ml | utilize freshly prepared |
| B. acetate buffer (pH 5.2) | a) 5.75 ml acetic acid and add deionized wate up to 1 L; b) 13.61 gr sodium acetate and add deionized water up to 1 L; mix a) and b) as following: 210 ml a) + 790 ml b) | RT |
| B. DNAse buffer | 1-100 mg/mL DNAse I, 30mM Trizma base (pH 7.2), 140mM K cacodylate, 4mM MgCl2, 0.1mM dithiothreitol | 0°C |
Appendix 2: Reagents
| 3-amino-9-ethylcarbazole (AEC) Sigma Aldrich | A5754 |
| anti-DNA-POD (clone MCA-33) Boehringer Mannhein | 1544675 |
| biotinylated dUTP Boehringer Mannhein | 1093070 |
| DAB tablets (100 tablets) Sigma Aldrich | D5905 |
| DNAse I Boehringer Mannhein | 776785 |
| Extravidin Peroxidase Sigma Aldrich | E2886 |
| N-N’-dimethylformamide Sigma Aldrich | D8654 |
| proteinase K Boehringer Mannhein | 745723 |
| serum albumin (BSA) Sigma Aldrich | B7276 |
| TdT enzyme Boehringer Mannhein | 220582 |
| Trizma base Sigma Aldrich | T6791 |
Appendix 3: Equipment
| Flow Cabinet TC60 | Gelaire |
| Incubator CO2-AUTO-ZERO | Heraeus |
| Microtome | Microm |
| Pipetman P20, P200, P1000 | Gilson |
Fig. 1. Immunocytochemical staining with anti-DNA-POD mAb. Mouse thymus apoptosis induced after in vivo treatment with dexametasone 21-phosphate. Stained apoptotic thymocytes (arrowheads, Fig. 1a) and macrophage with stained phagocytized apoptotic bodies in cytoplasm (arrowhead, Fig. 1b) are shown.
Bar = 10 mm.
