Lucassen P J, Goudsmit E, Pool C W, Mengod G, Palacios J M, Raadsheer F C, Guldenaar S E, Swaab D F
Netherlands Institute for Brain Research, Amsterdam, ZO.
J Neurosci Methods. 1995 Apr;57(2):221-30. doi: 10.1016/0165-0270(94)00152-7.
In order to study the suitability of formalin-fixed paraffin-embedded brain tissue for vasopressin (AVP)-mRNA detection, we used symmetric halves of 5 human hypothalami. In every case, one half was formalin fixed for 10-35 days and paraffin embedded while the other half was frozen rapidly. Following in situ hybridization (ISH) histochemistry on systematically obtained sections of the supraoptic (SON) and paraventricular nucleus (PVN) of both halves, total amounts of AVP-mRNA in these nuclei were estimated using densitometry of film autoradiographs. Total amounts of radioactivity were found to vary considerably between patients and amounted to 1297 +/- 302 arbitrary units (AU) (PVN) (mean +/- SEM) and 2539 +/- 346 (SON) for the cryostat sections and 868 +/- 94 (PVN) and 1259 +/- 126 (SON) for the paraffin tissue. Variations introduced by the method itself yielded a coefficient of variation of only 0.19. Furthermore, a non-significant negative trend with postmortem delay was found in cryostat tissue, but not in paraffin sections. No effect of fixation time was observed in the paraffin tissue. Both ways of tissue treatment have specific advantages and disadvantages that may be different for other probes or other brain areas. For ISH of a highly abundant mRNA like AVP in a very heterogeneous brain area such as the human hypothalamus, formalin-fixed paraffin-embedded tissue sections can be used for quantitative analysis of entire brain nuclei because of the small variation in this tissue, the remarkably good signal recovery (some 75% as compared to cryostat sections) and its practical advantages with regards to anatomical orientation, storage and sampling of the tissue.
为了研究福尔马林固定石蜡包埋的脑组织用于血管加压素（AVP）-mRNA检测的适用性，我们使用了5个人类下丘脑的对称两半。在每种情况下，一半用福尔马林固定10 - 35天并石蜡包埋，而另一半则快速冷冻。在对两半的视上核（SON）和室旁核（PVN）系统获取的切片进行原位杂交（ISH）组织化学后，使用胶片放射自显影片的光密度测定法估计这些核中AVP-mRNA的总量。发现患者之间放射性总量差异很大，对于冷冻切片，室旁核（PVN）为1297±302任意单位（AU）（平均值±标准误），视上核（SON）为2539±346；对于石蜡组织，室旁核（PVN）为868±94，视上核（SON）为1259±126。该方法本身引入的变异产生的变异系数仅为0.19。此外，在冷冻组织中发现了与死后延迟的非显著负相关趋势，但在石蜡切片中未发现。在石蜡组织中未观察到固定时间的影响。两种组织处理方式都有其特定的优缺点，对于其他探针或其他脑区可能不同。对于在人类下丘脑这样非常异质的脑区中对像AVP这样高度丰富的mRNA进行ISH，福尔马林固定石蜡包埋的组织切片可用于对整个脑核进行定量分析，因为该组织变异小、信号恢复非常好（与冷冻切片相比约为75%）以及在组织的解剖定位、储存和采样方面具有实际优势。
