Detection of apoptotic process in situ using immunocytochemical

1. INTRODUCTION 

• Apoptosis was observed from invertebrates to lower and higher verterbrates, and intervenes both in physiological and in pathological states, such as normal cell turnover, embryogenesis, metamorphosis, thymic maturation and involution, neoplasm, etc. (1-4).

Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. The typical "DNA ladder" on agarose gel electrophoresis, however, can not provide information regarding the histological localization at single cell level. This can be done by immunocytochemical method (A) or by enzymatic in situ labeling of apoptosis-induced DNA strand breaks (TUNEL) (B). 
 

A mouse anti-DNA monoclonal antibody (mAb) conjugated with peroxidase (anti-DNA-POD) was used. This anti-DNA-POD mAb binds to single- and double-stranded, low molecular weight DNA fragments (mono- and oligonucleosomes), showing the internucleosomal degradation of genomic DNA occuring during apoptosis. The tests were performed on sections of rat, chichen, frog and fish thymus. All the thymus were fixed in Bouin's fixative and embedded in paraffin wax (3).

The low molecular weight DNA fragments as well as single strand breaks ("nicks") in high molecular weight DNA can be identified by labeling free 3’-OH termini with modified nucleotides in an enzymatic reaction. Terminal deoxynucleotidyl transferase (TdT), which catalyzes polymerization of nucleotides to free 3’-OH DNA ends in a template-independent manner, is used to label DNA strand breaks. Incorporated nucleotides are detected by a secondary antibody, conjugated with peroxidase. After substrate reaction, stained cells can be detected under light microscope. Utilizing the incorporation of fluorescein-labeled nucleotides, the use of fluorescence microscopy is also possible.

2. PROTOCOLS 

A.2.1 Materials

Bouin's fixative (A1), alcohols, xylene, eukitt, Paraffin wax , anti-DNA-POD (A2), Phosphate Buffered Saline (PBS), 3,3'-diaminobenzidine tetrahydrochloride (DAB) tablets (A2), Phosphate-citrate buffer , Mayer's hematoxylin.

A.2.2. Methodology

• 1. Tissue sections are deparaffinated and bring up to 95% alcohol.

3. Inhibition of endogenous peroxidase 0.3% H

O

in methanol for 15 min.

4. Wash in running tap water for 10 min.

5. Wash in PBS (3x5 min).

6. Add anti-DNA-POD (1: 5; 1: 10) overnight at 4°C.

7. Wash in PBS (3x5 min).

8. Demonstration of peroxidase activity in a Petri dish: dissolve a DAB tablet (10 mg) in 40 ml of 0.1M citrate acid-sodium citrate buffer (pH 5.5), add 5 ml of H

O

and filter

9. Wash in running tap water for 5 min, deionized water.

10. Counterstain nuclei with Mayer's hematoxylin for 1 min.

11. Dehydrate through alcohols to xylene and mount in eukitt.

The immunocytochemical procedure described represents an original modification of the method for cell death detection by ELISA. Utilizing this new modified assay we are able to show the presence of apoptotic cells or cells involved in apoptosis in the thymus of all the species tested (Fig. 1). 
 

The results are confirmed with both negative and positive controls. The negative controls are performed by substituting the primary antibody with non-immune sera. The positive controls are performed by inducing in vitro and in vivo apoptosis and then visualized the phenomenon with anti-DNA mAb. In vitro experiments are performed by treating human lymphocytes with 2-deoxyribose and rat thymocytes with dexamethasone 21-phosphate. In vivo experiments are performed by injecting Swiss male albine mice with dexamethasone 21-phosphate.

The protocol require less than 90 min before the overnight incubation and 45 min after. 
 

         A.3.4 Key references

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.