University of Verona, Department of Neurosciences, Biomedicine and Movement Sciences, Verona, Italy.
University of Verona, Department of Neurosciences, Biomedicine and Movement Sciences, Verona, Italy.
Neurochem Int. 2021 Jan;142:104922. doi: 10.1016/j.neuint.2020.104922. Epub 2020 Nov 23.
Approaches utilizing multiple analysis techniques on a single sample are highly desirable in research, especially to reduce the number of animals and obtain the maximum information. Golgi-Cox staining is a widely used method for characterizing axon and dendritic morphology and several attempts to combine this technique with immunofluorescence and transmission electron microscopy have been proposed. With few exceptions, most of the protocols were characterized by a high degree of complexity and low reproducibility. Here we show a simplified procedure of perfusion, fixation and staining of brain tissues that allows Golgi-Cox staining, immunofluorescence and transmission electron microscopy in the same sample, to obtain high-quality images with a low-cost procedure. The main novelty in this protocol is the possibility of performing Golgi-Cox staining after the perfusion and post-fixation of brain tissue with a buffered solution containing, not only formaldehyde, but also glutaraldehyde. This renders the tissue suitable for electron microscopy, but it is also compatible with immunofluorescence staining. This combined protocol can be used in most neuroscience laboratories as it does not require special equipment and skills. This protocol will be useful in a broad range of neuroscience topics to study morphological changes during brain development and plasticity in physiological and pathological conditions.
在研究中，利用单一样本进行多种分析技术的方法是非常可取的，尤其是为了减少动物数量并获得最大信息量。高尔基-考克斯染色是一种广泛用于描述轴突和树突形态的方法，已经提出了几种将该技术与免疫荧光和透射电子显微镜结合的尝试。除了少数例外，大多数方案的特点是高度复杂且可重复性低。在这里，我们展示了一种简化的脑组织灌注、固定和染色程序，该程序允许在同一样本中进行高尔基-考克斯染色、免疫荧光和透射电子显微镜，从而以低成本获得高质量的图像。该方案的主要新颖之处在于，在含有甲醛和戊二醛的缓冲溶液中对脑组织进行灌注和后固定后，有可能进行高尔基-考克斯染色。这使得组织适合电子显微镜，但也与免疫荧光染色兼容。这种组合方案可在大多数神经科学实验室中使用，因为它不需要特殊设备和技能。该方案将在广泛的神经科学主题中有用，可用于研究脑发育和生理及病理条件下的可塑性过程中的形态变化。
