A SIMPLIFIED AND RAPID PROCEDURE FOR IN-SITU HYBRIDIZATION ON HUMAN, FLASH-FROZEN, POSTMORTEM BRAIN AND ITS COMBINATION WITH IMMUNOHISTOCHEMISTRY

A simplified and rapid method is described for in situ hybridization ( ISHH) studies of human post-mortem brain. Brain tissue was dissected i nto slices and was flash-frozen at -70 degrees C for storage. ISHH was carried out on 12 mu m cryostat sections, post-fixed in 4% paraformal dehyde. The histology of human brain tissue prepared by this technique rivalled that of formalin-fixed, wax-embedded tissue. In ISHH studies , flash-frozen tissue gave superior results to those obtained followin g long-term fixation of tissue in 10% formalin with subsequent wax-emb edding, or short-term prefixation in 4% paraformaldehyde. A systematic evaluation of commonly employed preparative procedures for ISHH was c arried out on flash-frozen brain and a simplified protocol, consisting only of fixation and dehydration, was developed as a result of these studies. Specific hybridization of probes to a number of mRNA species was demonstrable in neurons in different brain regions. Using 0.5% glu taraldehyde/4% paraformaldehyde post-fixation, immunohistochemical lab elling of TH-positive cortical catecholaminergic neurons and striatal dopaminergic terminals was successfully demonstrated in flash-frozen t issue. The same fixation technique also allowed combination of ISHH an d immunohistochemistry for the simultaneous demonstration of tyrosine hydroxylase mRNA and peptide in neurons of human brain stem and cortex . mRNA and peptides in flash-frozen tissue were found to be stable for more than 3 years. ISHH could be readily performed on relatively larg e brain structures. In addition to permitting excellent ISM and immuno histochemistry, alone or in combination, flash-freezing allows the max imum versatility of tissue use and does not compromise its study by ot her neuroscience techniques.