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Detection of apoptotic process in situ using immunocytochemical and TUNEL assays
Detection of apoptotic process in situ using immunocytochemical
and TUNEL assays
Daniela Barbieri * and Enzo Ottaviani °
 
*Dept. Biomedical Sciences, Sect. General Pathology, Via Campi, 287;
°Dept. Animal Biology, University of Modena, 41100 Modena, Italy
 

1. INTRODUCTION



   

2. PROTOCOLS



             A.3.4 Key references 1. Kerr, J.F.R., Wyllie, A.H., Currier, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer, 26: 239.

2. Ottaviani, E., Franchini, A. 1988. Ultrastructural study of haemocytes of the freshwater snail Planorbarius corneus (L.) (Gastropoda, Pulmonata) Acta Zool. (Stockh.) 69:157.

3. Ottaviani, E., Franchini, A., Franceschi, C. 1997. Evolution of neuroendocrine thymus: studies on POMC-derived peptides, cytokines and apoptosis in lower and higher vertebrates. J. Neuroimmunol. 72: 67.
 
4. Ueda, N., Shah, S.V. 1994. Apoptosis. J. Lab. Clin. Med. 124: 69.

B) TUNEL in situ procedure

 

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).

   
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) 
adjust pH at 5.2 

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.