Romijn H J, van Uum J F, Breedijk I, Emmering J, Radu I, Pool C W
Netherlands Institute for Brain Research, Amsterdam, The Netherlands.
J Histochem Cytochem. 1999 Feb;47(2):229-36. doi: 10.1177/002215549904700211.
The main goal of this study was to develop a better light microscopic procedure for quantitative study of the cellular co-localization of neuropeptides in adult human brain tissue. To reach this goal, we opted for a method (proved to be optimal on rat brain) in which sections were double immunolabeled with two different fluorophore-conjugated secondary antibodies and analyzed with a confocal laser scanning fluorescence microscope. One of our main problems faced was a strong autofluorescence of the sections, mainly caused by lipofuscin granules normally present in adult human brain tissue, which made any analysis of specific fluorescence impossible. This problem could be solved by staining the sections after immunolabeling with the dye Sudan Black B, which completely blocked this autofluorescence. The complete optimized procedure that we eventually developed can be summarized as follows. After a relatively short fixation time (6-14 days) in 4% freshly depolymerized paraformaldehyde, the resected brain tissue can best be stored in a 30% sucrose solution supplemented with 0.05% NaN3 at 4C. Stored under these conditions, cryosections from the tissue still reveal good histology and allow successful immunocytochemical staining after a period of 6 months. Double immunolabeling is done by incubating cryo- or paraffin sections in a mixture of two primary antibodies directed against the targeted antigens, followed by incubation with two different fluorophore-conjugated secondary antibodies. Amplification with a biotinylated secondary antibody followed by fluorophore-conjugated streptavidin is possible. Finally, the sections are stained with Sudan Black B, mounted in plain 80% Tris-buffered glycerol, and studied by confocal laser scanning fluorescence microscopy. Sections processed in this way are well suited for qualitative and quantitative analyses of co-localized neuropeptides in human brain tissue.
本研究的主要目标是开发一种更好的光学显微镜方法，用于定量研究成人大脑组织中神经肽的细胞共定位。为实现这一目标，我们选择了一种（已证明在大鼠脑上是最佳的）方法，即对切片用两种不同的荧光团偶联二抗进行双重免疫标记，并用共聚焦激光扫描荧光显微镜进行分析。我们面临的一个主要问题是切片的强烈自发荧光，这主要是由成人大脑组织中通常存在的脂褐素颗粒引起的，这使得对特定荧光的任何分析都无法进行。这个问题可以通过在用苏丹黑B染料进行免疫标记后对切片进行染色来解决，苏丹黑B完全阻断了这种自发荧光。我们最终开发的完整优化程序可总结如下。在4%新鲜解聚的多聚甲醛中固定相对较短的时间（6 - 14天）后，切除的脑组织最好保存在补充有0.05%叠氮化钠的30%蔗糖溶液中，于4℃保存。在这些条件下保存，组织的冷冻切片在6个月后仍能显示良好的组织学结构，并能成功进行免疫细胞化学染色。双重免疫标记是通过将冷冻或石蜡切片在针对目标抗原的两种一抗的混合物中孵育，然后与两种不同的荧光团偶联二抗孵育来完成的。可以用生物素化二抗随后用荧光团偶联的链霉亲和素进行放大。最后，切片用苏丹黑B染色，封固在普通的80% Tris缓冲甘油中，并用共聚焦激光扫描荧光显微镜进行研究。以这种方式处理的切片非常适合对人脑组织中共定位的神经肽进行定性和定量分析。
