Journal of Neuroscience Methods 189 (2010) 51–55 Contents lists available at ScienceDirect Journal of Neuroscience Methods journal homepage: www.elsevier.com/locate/jneumeth The “single-section” Golgi method adapted for formalin-fixed human brain and light microscopy Aline Dall’Oglio a,1 , Denise Ferme b,1 , Janaína Brusco c,d , Jorge E. Moreira c,d , Alberto A. Rasia-Filho a,b,e,∗ a Program in Neuroscience, Institute of Basic Sciences, Federal University of Rio Grande do Sul, R. Sarmento Leite 500, Porto Alegre RS 90050-110, Brazil b Program in Pathology, Federal University of Health Sciences, R. Sarmento Leite 245, Porto Alegre RS 90050-110, Brazil c Laboratory of Synaptic Structure, Department of Cell/Molecular Biology and Biopathogens, Medical School of Ribeirão Preto, University of São Paulo, Av. Bandeirantes 3900, Ribeirão Preto SP 14049-900, Brazil d Program in Neuroscience & Behavior, Ribeirão Preto School of Medicine, Universidade de São Paulo, Brazil e Department of Basic Sciences/Physiology, Federal University of Health Sciences, R. Sarmento Leite 245, Porto Alegre RS 90050-110, Brazil article info Article history: Received 26 November 2009 Received in revised form 15 March 2010 Accepted 16 March 2010 Keywords: Golgi method Neuronal morphology Glial cells abstract The Golgi method has been used for over a century to describe the general morphology of neurons in the nervous system of different species. The “single-section” Golgi method of Gabbott and Somogyi (1984) and the modifications made by Izzo et al. (1987) are able to produce consistent results. Here, we describe procedures to show cortical and subcortical neurons of human brains immersed in formalin for months or even years. The tissue was sliced with a vibratome, post-fixed in a combination of paraformaldehyde and picric acid in phosphate buffer, followed by osmium tetroxide and potassium dicromate, “sandwiched” between cover slips, and immersed in silver nitrate. The whole procedure takes between 5 and 11 days to achieve good results. The Golgi method has its characteristic pitfalls but, with this procedure, neurons and glia appear well-impregnated, allowing qualitative and quantitative studies under light microscopy. This contribution adds to the basic techniques for the study of human nervous tissue with the same advantages described for the “single-section” Golgi method in other species; it is easy and fast, requires minimal equipment, and provides consistent results. © 2010 Elsevier B.V. All rights reserved. 1. Introduction The colorazione nera has been used to reveal the general mor- phology of neurons and glia in various areas of the nervous system of several species (Golgi, 1873; Ramón y Cajal, 1909; Valverde, 1962; Fairén et al., 1977; Scheibel and Scheibel, 1978; McDonald, 1982). For more than a century, phylogenetic and ontogenetic stud- ies have been benefited from the Golgi method, which is a reliable tool to identify different types of neurons, the detailed shape of single cells, and to extract functional hypotheses of brain func- tion from morphology (Ramón y Cajal, 1909; Lorente de Nó, 1934; Valverde, 1962; Szentágothai, 1978; Kisvárday et al., 1990; Larriva- Sahd, 2008; Gómez-Villalobos et al., 2009). As summarized by Fairén (2005), the Golgi method is based on the metallic impregnation of neurons and glial cells in tissue blocks that had been hardened by potassium dichromate and then treated by silver nitrate. A proper historical and technical review of the diverse Golgi procedures can be found in Alonso (1994) and refer- ∗ Corresponding author at: UFCSPA/Department of Basic Sciences, R. Sarmento Leite 245 (room 308), Porto Alegre 90170-050 RS, Brazil. Tel.: +55 51 91161643; fax: +55 51 33038752. E-mail addresses: rasiafilho@pq.cnpq.br, aarf@ufcspa.edu.br (A.A. Rasia-Filho). 1 Both authors contributed equally to this study. ences therein (see also Nauta and Ebbesson, 1970; Banks, 1999). Following the original description of his “slow method”, which used a sequence of potassium or ammonium dichromate and silver nitrate for long time, Golgi proposed that a potassium dichromate and osmium tetroxide mixture could speed up the reaction. This “rapid method” was further developed and masterly applied by Ramón y Cajal. Golgi also tested the use of mercury salts during tis- sue fixation and for cellular impregnation, which Cox successfully modified later. Other efforts involved the use of different aldehydes as alternative fixative solutions and generated the Golgi–Kopsch, the Golgi–Colonnier, and the Golgi–Rio Hortega procedures, to cite some of them (Alonso, 1994). After a period of oblivion until the sec- ond half of the 20th century (Fairén, 2005), Golgi method revived (e.g., Valverde, 1970; Fairén et al., 1977, 1984; Kolb et al., 1981; McDonald, 1982; Feldman, 1984; Millhouse, 1986; Somogyi, 1990; Woolley et al., 1990; Woolley and McEwen, 1994; Jacobs et al., 1997; Dall’Oglio et al., 2008b; Larriva-Sahd, 2008). All of the procedures in the “family” of Golgi techniques have advantages and restrictions. For example, silver impregnation apparently occurs “at random” and there are no unequivocal expla- nations about discrepant results of the method. This leads to a number of impregnated neurons but not a homogeneous pattern of cellular staining on each tissue section. To provide useful results, it has been controled the diffusion rate and the pH of the chromation 0165-0270/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2010.03.018