Louth Emma L, Sutton Charles D, Mendell Ari L, MacLusky Neil J, Bailey Craig D C
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph.
Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph;
J Vis Exp. 2017 Apr 18(122):55358. doi: 10.3791/55358.
The Golgi-Cox method of neuron staining has been employed for more than two hundred years to advance our understanding of neuron morphology within histological brain samples. While it is preferable from a practical perspective to prepare brain sections at the greatest thickness possible, in order to increase the probability of identifying stained neurons that are fully contained within single sections, this approach is limited from a technical perspective by the working distance of high-magnification microscope objectives. We report here a protocol to stain neurons using the Golgi-Cox method in mouse brain sections that are cut at 500 μm thickness, and to visualize neurons throughout the depth of these sections using an upright microscope fitted with a high-resolution 30X 1.05 N.A. silicone oil-immersion objective that has an 800 μm working distance. We also report two useful variants of this protocol that may be employed to counterstain the surface of mounted brain sections with the cresyl violet Nissl stain, or to freeze whole brains for long-term storage prior to sectioning and final processing. The main protocol and its two variants produce stained thick brain sections, throughout which full neuron dendritic trees and dendrite spines may be reliably visualized and quantified.
高尔基-考克斯神经元染色法已被应用了两百多年，以增进我们对组织学脑样本中神经元形态的理解。从实际角度来看，为了提高识别完全包含在单个切片内的染色神经元的概率，尽可能制备最厚的脑切片是可取的，但从技术角度来看，这种方法受到高倍显微镜物镜工作距离的限制。我们在此报告一种方案，使用高尔基-考克斯方法对厚度为500μm的小鼠脑切片中的神经元进行染色，并使用配备有工作距离为800μm的高分辨率30X 1.05 N.A.硅油浸液物镜的直立显微镜观察这些切片整个深度的神经元。我们还报告了该方案的两个有用变体，可用于用甲酚紫尼氏染色对 mounted脑切片表面进行复染，或在切片和最终处理之前冷冻整个大脑以进行长期储存。主要方案及其两个变体可产生染色的厚脑切片，在整个切片中可以可靠地观察和量化完整的神经元树突和树突棘。
