A technique for the electron microscopic autoradiography of Al adenosine receptors in brain tissue

Summary A procedure for the electron microscopic autoradiography of Al adenosine receptors is described. Fresh tissue slices from rat hippocampus were incubated with the radioactive adenosine analogs: Cyclohexyl³H]adenosine, 5-N-ethylcarboxamido³H]adenosine or ¹²⁵I]-iodohydroxyphenylisopropyladenosine. Various fixation agents were tested with respect to the retention of these ligands by the tissue. While most of the ligands were lost in aldehyde fixation they were retained by osmium tetroxide probably via a crosslinking reaction. The final method of choice was an aldehyde prefixation (in the case of ¹²⁵I]-iodohydroxyphenylisopropyladenosine with 4% buffered paraformaldehyde) during which more than 90% of the nonspecifically bound ligands were washed out while 40% of the specifically bound ligands remained. Subsequent fixation with osmium tetroxide (1%) allowed a standard protocoll for dehydration and embedding to be used with only minimal (less than 5%) further loss of the ligands. Electron microscopic autoradiography provided evidence for a specific distribution of the binding sites for ¹²⁵I]-iodohydroxyphenylisopropyladenosine.Abbreviations GA Glutardialdehyde - PFA Paraformaldehyde - OsO₄ Osmiumtetroxide - CHA Cyclohexyladenosine - NECA N-ethyl-carboxamidoadenosine - PIA Phenylisopropyladenosine - I-HPIA Iodohydroxyphenylisopropyladenosine - HPIA Hydroxyphenylisopropyladenosine