Simultaneous Golgi-Cox and immunofluorescence using confocal microscopy

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.