Simultaneous Golgi-Cox and immunofluorescence using confocal microscopy

Abstract

Visualization of neuronal elements is of fundamental importance in modern neuroscience. Golgi-Cox impregnation is a widely employed method that provides detailed information about morphological characteristics of neurons, but none regarding their neurochemical features. Immunocytochemical procedures, on the other hand, can provide a high degree of biochemical specificity but poorer morphological details, in particular if compared to Golgi-Cox impregnation. Hence, the combined use of these two approaches is highly desirable, especially for confocal microscopy that can exploit the advantages of both methods simultaneously. Here we show an innovative procedure of perfusion and fixation of brain tissue, that allows, by applying Golgi-Cox impregnation and immunofluorescence in the same histological section, to obtain high-quality histological material, with a very simple and inexpensive method. This procedure is based on three simple fixation steps: (1) a paraformaldehyde perfusion followed by a standard post-fixation to stabilize the subsequent immunofluorescence reaction; (2) the classical Golgi-Cox impregnation and (3) an immunofluorescence reaction in previously impregnated material. This combination allows simultaneous visualization of (a) the structural details (Golgi-Cox impregnated neurons), (b) the antigens’ characterization, (c) the anatomical interactions between discrete neuronal elements and (d) the 3D reconstruction and modeling. The method is easy to perform and can be reproducibly applied by small laboratories and expanded through the use of different antibodies. Overall, the method presented in this study offers an innovative and powerful approach to study the nervous system, especially by using confocal microscopy.

Comprehensive Review of Golgi Staining Methods for Nervous Tissue

“…However, as with the other staining methods, there is the disadvantage that neurons are randomly stained. To enhance the specificity of staining, a method that combined immunofluorescence and Golgi staining was recently developed (Spiga et al, 2011).…”

“…Because the background noise is low, it is better to use this staining method to clearly observe dendritic spines and cell bodies. Several methods have been recently attempted to complement the nonspecific Golgi-Cox method, one of which is the simultaneous Golgi-Cox and immunofluorescence method by Spiga et al (2011) (Koyama & Tohyama, 2013). By simultaneously staining brains using immunofluorescence and the Golgi-Cox method, neurons can be observed by targeting specific areas.…”

“…Thus, there are approximately 3000 publications that used Golgi staining between 1965 and1999. While Golgi staining is continues to be used as a central technique, it has been combined with fluorescent staining (Koyama & Tohyama, 2013;Spiga et al, 2011). In addition, the latest developments in 3D Edge microscopy provide a solution to overcome the limitation of focal depth during the analysis of thick specimens (Rhyu et al, 2016).…”

A half century of experimental neuroanatomical tracing

“…Via post-acquisition computer image processing (deconvolution and rendering methods), the background ‗noise' is greatly reduced and an unprecedented level of rendering is obtained. Most importantly, a procedure enabling the combination of Golgi-impregnated material with immunofluorescence processing to reveal a number of neuronal markers has been developed very recently (Spiga et al, 2011). Examples of Golgi-Cox impregnated material as seen under the confocal microscope are illustrated in Figure 9.…”

Recent Progress of the Golgi Technique and Electron Microscopy To Examine Dendritic Pathology In Alzheimer‘s Disease

“…In a recent study on a diabetic rat model, swelling of axons and varicosities of dendrites were described in the cortex, associated with hyperphosphorylated tau, also seen at the early stages of Alzheimer's disease [17]. The visualization of dendrites and dendritic spines by the high-quality Golgi-Cox technique, in combination with immunocytochemical procedures, viewing in confocal microscopy, is a method of fundamental importance in neuroscience, since it allows simultaneous appearance of the dendritic arborization and its antigens' characterization, in a 3D way, facilitating multiple analyses in experimental models [18].…”