A simplified and rapid procedure for in situ hybridization on human, flash-frozen, post-mortem brain and its combination with immunohistochemistry

Journal of Neuroscience Methods
A E KingsburyO J Foster

Abstract

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 combin...Continue Reading

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Citations

Aug 5, 2000·Brain Research. Molecular Brain Research·Z Y TongO J Foster
Dec 5, 1998·Brain Research. Molecular Brain Research·D J EveO J Foster
Jul 13, 2002·Brain Research. Brain Research Protocols·Sathyanesan Samuel NewtonRonald Duman
Oct 1, 2013·Lancet Neurology·Neshika SamarasekeraJames W Ironside
Feb 24, 2004·Movement Disorders : Official Journal of the Movement Disorder Society·Ann E KingsburyOliver J F Foster
Mar 22, 2000·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·H LouisJ M Lamazière
Mar 26, 1999·Movement Disorders : Official Journal of the Movement Disorder Society·A E KingsburyO J Foster
Feb 14, 2006·Neurochemical Research·Sergio MontesPablo Muriel
May 5, 2006·Neurochemical Research·B GulyásG Székely

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