A simplified and rapid procedure for in situ hybridization on human, flash-frozen, post-mortem 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 into slices and was flash-frozen at -70 degrees C for storage. ISHH was carried out on 12 microns cryostat sections, post-fixed in 4% paraformaldehyde. 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 following long-term fixation of tissue in 10% formalin with subsequent wax-embedding, or short-term prefixation in 4% paraformaldehyde. A systematic evaluation of commonly employed preparative procedures for ISHH was carried 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% glutaraldehyde/4% paraformaldehyde post-fixation, immunohistochemical labelling of TH-positive cortical catecholaminergic neurons and striatal dopaminergic terminals was successfully demonstrated in flash-frozen tissue. The same fixation technique also allowed combination of ISHH and 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 large brain structures. In addition to permitting excellent ISHH and immunohistochemistry, alone or in combination, flash-freezing allows the maximum versatility of tissue use and does not compromise its study by other neuroscience techniques.
Kingsbury
A E
AE
Parkinson's Disease Society Brain Bank, Institute of Neurology, London, UK.
Bray
E L
EL
Foster
O J
OJ
D000368:Aged; D000369:Aged, 80 and over; D001345:Autoradiography; D001933:Brain Stem / Q000473:pathology*; D002540:Cerebral Cortex / Q000473:pathology*; D005260:Female; D005615:Freezing; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D017403:In Situ Hybridization / Q000379:methods*; D008297:Male; D008867:Microtomy; D009474:Neurons / Q000737:chemistry / Q000201:enzymology; D015345:Oligonucleotide Probes; D012333:RNA, Messenger / Q000032:analysis; D012680:Sensitivity and Specificity; D016707:Tissue Fixation / Q000592:standards; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D014446:Tyrosine 3-Monooxygenase