Proper visualization of neuronal elements is of fundamental importance in modern neuroscience. The Golgi-Cox method, since its discovery, has been usefully applied for qualitative and quantitative analysis of neuronal morphology. This method is a widely employed and provides detailed information about morphological characteristics of neurons, but none regarding their neurochemical features. Instead, immunohistochemical procedures, due to the ability of antibodies to bind specific antigens with high affinity, can provide a high degree of biochemical specificity but with less morphological detail if compared to Golgi-Cox impregnation. Hence, the combined use of these two approaches is highly desirable especially for confocal users 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 i) structural details (Golgi-Cox impregnated neurons), ii) antigens' characterization, iii) morphological-neurochemical interactions between discrete neuronal elements iv) 3-D reconstruction and modeling v) and reduction of animals required. 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.
Simultaneous Golgi-Cox and immunofluorescence labeling in brain slices by confocal microscopy
SPIGA, SATURNINO;
2011-01-01
Abstract
Proper visualization of neuronal elements is of fundamental importance in modern neuroscience. The Golgi-Cox method, since its discovery, has been usefully applied for qualitative and quantitative analysis of neuronal morphology. This method is a widely employed and provides detailed information about morphological characteristics of neurons, but none regarding their neurochemical features. Instead, immunohistochemical procedures, due to the ability of antibodies to bind specific antigens with high affinity, can provide a high degree of biochemical specificity but with less morphological detail if compared to Golgi-Cox impregnation. Hence, the combined use of these two approaches is highly desirable especially for confocal users 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 i) structural details (Golgi-Cox impregnated neurons), ii) antigens' characterization, iii) morphological-neurochemical interactions between discrete neuronal elements iv) 3-D reconstruction and modeling v) and reduction of animals required. 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.
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