title: 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.
authors: Department of Animal Biology and Ecology, University of Cagliari, Via Ing. Tomaso Fiorelli, 09126 Cagliari, Italy, sspiga@unica.it
authors: Saturnino
authors: S
authors: Spiga
authors: Elio
authors: E
authors: Acquas
authors: Maria C
authors: MC
authors: Puddu
authors: Giovanna
authors: G
authors: Mulas
authors: Alessandra
authors: A
authors: Lintas
authors: Marco
authors: M
authors: Diana
mesh_terms: D000818:Animals; D002840:Chromates; D000076122:Disks Large Homolog 4 Protein; D005455:Fluorescent Antibody Technique / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins; D008627:Mercuric Chloride; D018613:Microscopy, Confocal / Q000379:methods*; D008867:Microtomy / Q000379:methods; D009474:Neurons / Q000166:cytology* / Q000378:metabolism*; D017680:Potassium Compounds; D011192:Potassium Dichromate; D051381:Rats; D017207:Rats, Sprague-Dawley; D013194:Staining and Labeling / Q000379:methods*; D016704:Synapsins; D014446:Tyrosine 3-Monooxygenase
chemical_list: D002840:Chromates; D000076122:Disks Large Homolog 4 Protein; C501449:Dlg4 protein, rat; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D017680:Potassium Compounds; D016704:Synapsins; D008627:Mercuric Chloride; C027373:potassium chromate(VI); D014446:Tyrosine 3-Monooxygenase; D011192:Potassium Dichromate