Radioimmunoassay of met-enkephalin in microdissected areas of paraformaldehyde-fixed rat brain.
We studied the effect of various sample preparation procedures on rat brain met-enkephalin content, measured by radioimmunoassay. Whole brain met-enkephalin content of rats killed by decapitation followed by immediate tissue freezing was similar to that of rats killed by microwave irradiation and to those of rats anesthetized with pentobarbital or halothane before killing, whether previously perfused with paraformaldehyde or not. In contrast, a decrease (up to 80%) in met-enkephalin concentrations was observed when brain samples were frozen and thawed to mimic the procedure utilized in the "punch" technique for analysis of discrete brain nuclei. This decrease was totally prevented by paraformaldehyde perfusion of the brain prior to sacrifice. Brain perfusion did not alter the amount of immunoassayable met-enkephalin extracted from tissue or its profile after Sephadex chromatography. Paraformaldehyde perfusion results in better morphological tissue preservation and facilitates the "punch" dissecting technique. Paraformaldehyde perfusion may be the procedure of choice for the measurement of neuropeptides in specific brain nuclei dissected by the "punch" technique.
D000758:Anesthesia; D000818:Animals; D001923:Brain Chemistry; D001933:Brain Stem / Q000032:analysis; D003342:Corpus Striatum / Q000032:analysis; D004744:Enkephalin, Methionine / Q000032:analysis*; D005404:Fixatives; D005557:Formaldehyde; D005615:Freezing; D006624:Hippocampus / Q000032:analysis; D007031:Hypothalamus / Q000032:analysis; D008297:Male; D008872:Microwaves; D009419:Nerve Tissue Proteins / Q000032:analysis; D010477:Perfusion; D011108:Polymers; D011180:Postmortem Changes; D011863:Radioimmunoassay; D051381:Rats; D011919:Rats, Inbred Strains; D013048:Specimen Handling / Q000379:methods
D005404:Fixatives; D009419:Nerve Tissue Proteins; D011108:Polymers; D005557:Formaldehyde; D004744:Enkephalin, Methionine; C003043:paraform