Histochemical technique for the detection of chloride cells in fish Bruno Fiorelini Pereira a, *, Fla ´ vio Henrique Caetano b a Departamento de Biologia, Universidade Estadual Paulista, CEP: 1306558, Rio Claro, SP, Brazil b Departamento de Biologia, Universidade Estadual Paulista, CEP: 13506900, Rio Claro, SP, Brazil 1. Introduction Fish gills play vital roles, as they are the main site of gaseous exchanges, and as are involved in osmoregulation, acid–base balance, excretion of nitrogen compounds, and tasting (Machado, 1999). According to Karnaky et al. (1976a) it is well known that the most important osmoregulatory organ in fish is the gill, which is characterized by a relatively slow absorption of Na and C1 in low salinity environments, fresh water, and a relatively rapid secretion of Na and C1 in high salinity environments, seawater. The gill epithelium is stratified and consists of several cell types, including pavement cells, mucus cells, and chloride cells, in addition to taste buds and undifferentiated supporting cells (Machado, 1999; Garcia-Santos et al., 2007). According to Perry (1997), chloride cells were first identified by Keys and Wilmer (1932), while describing mitochondria-rich cells responsible for the secretion of Cl À in seawater adapted teleosts. Chloride cells are large, scattered in the lamellar surface, rich in mitochondria and participate in the process of active transport of ions (Machado, 1999; Perry, 1997). According to Evans (2008), since early physiological studies, several groups have demonstrated that chloride cells of teleosts express Na–K-activated ATPase. Because of the large number of mitochondria present, they are also know as mitochondria-rich cells or MRC. According to Perry (1997), studies suggest that chloride cells are the site of active ion transporting processes involved in ionic regulation in marine and freshwater fishes. This regulation, especially in marine fish, can account for a substantial portion of the energy budget, due to the hydrolysis of ATP by the various ı ´on-transport ATPases. These include Na + /K + -ATPase, high-affinity Ca 2+ -ATPase, and perhaps the poorly defined Cl À /HCO 3À -ATPase. The chloride cells are probably an important site of NaCl uptake in fresh water adapted fish and therefore chloride cells proliferation in low-[NaCl] water may be an important mechanism contributing to the enhance- ment of gill NaCl transport capacity. According to Karnaky et al. (1976b) the chloride cells are larger and/or more numerous in sea water than in fresh water fish and the largest cells of all occur in fish adapted to environments more concentrated than sea water. Under unfavorable ionic conditions or in contact with toxic agents, chloride cells proliferate in the filaments and on the lamellar surface of the gill (Perry, 1997) and according to Perry and Laurent (1989) the most important studies made in this area are related with the physiological and biochemical adaptation adjustments associated with euryhalinity. According to Perry and Walsh (1989), chloride cells have higher metabolic rates than those of other gill cells and the metabolic rate of the remaining of the gill directly affects the population of chloride cells, which may induce their proliferation. Micron 40 (2009) 783–786 ARTICLE INFO Article history: Received 28 May 2009 Received in revised form 7 July 2009 Accepted 16 July 2009 Keywords: Chloride cells Fish Von Kossa Gill ABSTRACT Chloride cells are responsible for ionic exchanges in the fish gill. These cells have been widely studied, considering its importance in vital functions of the gill, and because they proliferate when exposed to unfavorable environments. One of the main characteristic of these cells is an acidic cytoplasm, which has been used for identification through histochemical techniques with dyes such as Toluidine Blue and Hematoxylin and Eosin. However, these techniques can be problematic, since epithelial cells can, in certain situations, acquire acidic characteristics similar to those of chloride cells, thus staining in a similar way. Among other functions, chloride cells play a role in calcium uptake from the environment, and therefore have a high concentration of this element. Based on this information, this study aims at developing a specific protocol for the identification of chloride cells. With this purpose, the Von Kossa method specific for calcium was used combined with Hematoxylin counterstaining. Chloride cells had cytoplasm slightly stained with Hematoxylin and the presence of dark stained granules dispersed in the cytoplasm resulted from the Von Kossa reaction due to the calcium present in these cells. This was not found in any other gill cell. Thus, the technique used in this study was specific and efficient to identify chloride cells in fish gills. ß 2009 Elsevier Ltd. All rights reserved. * Corresponding author. E-mail addresses: brunofp22@hotmail.com (B.F. Pereira), fcaetano@rc.unesp.br (F.H. Caetano). Contents lists available at ScienceDirect Micron journal homepage: www.elsevier.com/locate/micron 0968-4328/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2009.07.010