Kingsbury A E, Bray E L, Foster O J
Parkinson's Disease Society Brain Bank, Institute of Neurology, London, UK.
J Neurosci Methods. 1996 Nov;69(2):213-27. doi: 10.1016/S0165-0270(96)00086-6.
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.
本文描述了一种用于人类死后大脑原位杂交（ISHH）研究的简化快速方法。将脑组织切成薄片，在-70℃下速冻保存。ISHH在12微米的低温切片上进行，切片后用4%多聚甲醛固定。用该技术制备的人类脑组织的组织学与福尔马林固定、石蜡包埋的组织相当。在ISHH研究中，速冻组织的效果优于长期用10%福尔马林固定并随后石蜡包埋的组织，或短期用4%多聚甲醛预固定的组织。对速冻大脑进行了ISHH常用制备程序的系统评估，并基于这些研究开发了一种仅由固定和脱水组成的简化方案。在不同脑区的神经元中可证明探针与多种mRNA种类的特异性杂交。使用0.5%戊二醛/4%多聚甲醛后固定，在速冻组织中成功显示了TH阳性皮质儿茶酚胺能神经元和纹状体多巴胺能终末的免疫组织化学标记。相同的固定技术还允许将ISHH和免疫组织化学结合，同时显示人脑干和皮质神经元中的酪氨酸羟化酶mRNA和肽。发现速冻组织中的mRNA和肽可稳定保存3年以上。ISHH可在相对较大的脑结构上轻松进行。除了单独或联合允许进行出色的ISHH和免疫组织化学外，速冻还能使组织的使用具有最大的通用性，并且不会影响其他神经科学技术对其进行研究。
