New Tetrachromic VOF Stain (Type III-G.S) for Normal and Pathological Fish Tissues C
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ORIGINAL PAPER New Tetrachromic VOF Stain (Type III-G.S) for Normal and Pathological Fish Tissues C. Sarasquete,* M. Gutiérrez Instituto de Ciencias Marinas de Andalucía, CSIC Polígono Río San Pedro, Apdo oficial, Puerto Real, Cádiz, Spain richrome methods invariably use dyes in acid ©2005, European Journal of Histochemistry pH solvents, usually diluted in aqueous acetic Tacid, and the concentration of this acid A new VOF Type III-G.S stain was applied to histological sec- matches the concentration of dye. Staining depends tions of different organs and tissues of healthy and pathologi- largely on the attachment of dyes to proteins. The cal larvae, juvenile and adult fish species (Solea senegalensis; acid pH itself is necessary to maximise the amount Sparus aurata; Diplodus sargo; Pagrus auriga; Argyrosomus regius and Halobatrachus didactylus). In comparison to the of dye that will attach to tissue amino groups. original Gutiérrez´VOF stain, more acid dyes of contrasting Proteins have both positively (amino groups) and colours and polychromatic/metachromatic properties were negatively (carboxyl and hydroxyl) charged groups. incorporated as essential constituents of the tetrachromic VOF Usually one predominates and this will have an stain. This facilitates the selective staining of different basic tissues and improves the morphological analysis of histo- overall negative or positive charge (being an acid or chemical approaches of the cell components. The VOF-Type III a basic protein). These charges can, however, bal- G.S stain is composed of a mixture of several dyes of varying ance each other out to some degree. Phosphate size and molecular weight (Orange G< acid Fuchsin< Light green<Methyl Blue<Fast Green), which are used simultane- groups of DNA and binding-proteins are important ously, and it enables the individual tissues to be selectively dif- in nuclear staining.The ionisation of basic groups of ferentiated and stained. Muscle fibers, collagen, reticulin and proteins predominates at acid pH, and correspon- elastin fibers, erythrocytes, cartilage, bone, mucous cells, oocytes and larvae were selectively stained and differentiated. ding tissues will have affinity to acid dyes Dyes with small size and molecular weight (i.e Orange G), pen- (Gutiérrez, 1990). In addition, dyes also have the etrate all tissue structures rapidly, but are only tightly retained same groups as the proteins, but may include the in densely textured tissues (i.e erythrocytes). Methyl Blue is an sulphonic group as well. Which of these groups is interesting triarylmethane dye (large size and molecular weight), which is incorporated in this new VOF tetrachrome involved in any particular case depends on the cir- stain, and acquires histochemical significance when used at cumstances, including the pH of the staining solu- acid pH (2.8) because collagen and reticulin fibers, as well tion (Culling, 1976; Lillie, 1977; Pearse, 1985; basophilic and metachromatic substances (strongly ionized sulphated glycoconjugates) can be identified. Muscle tissues Gutiérrez, 1990; Kiernan, 1999). show an evident green colour (Fast Green or Light Green affini- Trichrome stains can be applied as a one-step ties), even those isolated and/or diffuse muscle fibers pres- technique (i.e. van Gieson’s and Gomori’s methods) ent in the digestive submucosa layer. Connective tissues showed a specific and strong blue colour (Methyl Blue affini- or as a multi-step method (i.e Masson's trichrome), ty) or mixed blue-red staining (Methyl Blue and Acid Fucshin where dyes and reagents are applied sequentially, affinities). Very noticeable is the staining of the mucous cells, and staining is optimised at each step (Culling, as well as the hyaline capsule of the viral lymphocystic cells, 1976; Lillie, 1977, Pearse, 1985; Bancroft and which were stained blue-purple (carboxylated and/or strongly ionized sulphated groups). Cartilaginous tissues showed a Cook, 1984; Gutiérrez, 1990; Kiernan, 1999). blue or purple (Methyl Blue affinity) staining, and a specific The original Haematoxylin-Gutiérrez´VOF stain red colour (Acid Fucshin affinity) was evident during calcifica- (Gutierrez, 1961,1967) is a one-step trichromic tion or in bone structures (i.e skeleton, fins, gills, teeth). stain, useful for histological purposes and Key words: Tetrachromic stain, cartilage, bone, fibers, diges- histopathological diagnosis (Gutiérrez et al., 1963; tive tract, ovary, skin, teeth, fish species. Gutiérrez, 1990). Specially interesting is the matu- *Correspondence: [email protected] Tel: 34.956832612. ration of the protozoan parasite, Martelia refrigens Fax: 34.956-834701. detected in the digestive gland of mussels, Mytilus Paper accepted on January 07, 2005 edulis, by using the original Haematoxylin- VOF stain (Gutiérrez, 1977a, b), because mature stages European Journal of Histochemistry 2005; vol. 49 issue 2 (Apr-Jun): 105-114 of this parasite were clearly evidenced by a specific Orange G affinity, while inmature stages stain blue 105 C. Sarasquete et al. due to a specific Haematoxylin affinity. Also, aci- dophilic zymogen granules secreted by the Material and Methods basophilic exocrine pancreas, erythrocytes accumu- lated in vascular systems, and yolk (larvae and Fixation and embedding samples oocytes) showed a strong or specific affinity to Samples (8-15x3-4 mm) of different tissues Orange G dye (Gutiérrez et al., 1985; Sarasquete et (ovary, digestive tract, gills, kidney, spleen, liver, al., 1993, 1995, 2002; Ribeiro et al., 1999; Gisbert heart) of healthy and pathological organisms (viral et al., 1999; Ortiz-Delgado et al., 2003; infected skin) and whole larvae and juvenile speci- Santamaria et al., 2004). On the other hand, and mens (Senegales sole, Solea senegalensis; interestingly, the mucous secreting cells containing seabream, Sparus aurata; sargo, Diplodus sargo; neutral and/or carboxylate- and/or sulphate-rich redbanded seabream, Pagrus auriga; meagre, glycoconjugates are present in the digestive tract, Argyrosomus regius and toadfish, Halobatrachus gills and skin of different larvae and adult fish didactylus) are fixed in Helly´s or Bouin´s fluids species (Gutiérrez et al., 1986; Sarasquete et al., during 2-10 h, depending on their thickness. Most 1995, 1996, Gisbert et al., 1999; Ribeiro et al., trichrome stains benefit from picric acid or mer- 1999, Ortiz-Delgado et al., 2003; Arellano et al., curic chloride fixation. If tissues have been fixed in 2001, 2004). Usually, these secretive cells are PAS formalin 10% buffered with phosphate (0.1 M at and Alcian Blue positive, but they are unstained pH 7.2), fixed sections can be postfixed in Bouin's with Haematoxylin-eosin or Haematoxylin- fluid or treated in picro-mercuric-alcohol (a satu- Gutiérrez´VOF morphological techniques rated solution of picric acid in absolute alcohol (Sarasquete et al., 1995, 2001; Arellano et al., containing 3% mercuric chloride) overnight. 2001, 2002, 2004; Ortiz-Delgado et al., 2003). Subsequently, mercuric pigment must be removed Pituitary glands of vertebrates have been largely with iodine/thiosulphate and the sections washed in used for researching trichromic stains. In fact, by water to remove picric acid. After fixation, samples using both Gutiérrez´VOF stain (Gutiérrez, 1961, are washed in 80% ethanol, and dehydrated 1967, 1990) and Slidders´method (Slidders, through increasing ethanol series (or acetone), 1961), which are composed of three (Light Green, cleared and embedded in 56-60ºC paraffin. Orange G and Acid Fucshin) isolated or mixed dyes, Sections are routinely made at 4-6 µm, stained and pituitary glands of vertebrates present an interest- mounted in Eukit medium. ing colour variability of hormone-secreting cells; Several triarylmethane derivatives: Light Green showing the acidophilic cells as orange or green; the SF (C37H34N2O9S3Na2, MW 792.875)/or Fast basophilic cells as magenta-red and the chromo- Green FCF (C37H34N2O10S3Na2, MW 808.86); phobic/amphiphilic cells as pale grey or green Methyl Blue (C37H27N3O9S3Na2, MW 799.81) and (Slidders, 1961; Rendón et al., 1997; Sarasquete et Acid Fuchsin (C20H17N3O9S3Na2, MW 585.55) al., 1997; Rodríguez-Gómez et al., 2001). The and a monoazoic dye: Orange G (C16H10N2O7S2Na2, architectural pattern of pituitary or stroma of dif- MW 452.386) are used to prepare the original ferent organs/tissues (Culling, 1976; Lillie, 1977; VOF´Gutiérrez, VOF Type-II and VOF Type III G.S Rendon et al., 1997) is well demonstrated with stains (For review Lillie, 1977 and Zollinger,1991; reticulin stains (i.e Methyl Blue); and this triaryl- http://members.pgonline.com/~bryand/StainsFile/d methane (large size and molecular weight) and yes/). polychromatic dye (positive and negative charges) Tetrachromic VOF Type III -G.S stain solution is an essential component of the now proposed 1. Dissolve Fast Green FCF (260 mg) or Light tetrachromic VOF Type III -G.S stain. Green SF (260 mg), Methyl Blue (140 mg), In this paper we described a variant of the origi- Orange G (500 mg) and Acid Fuchsin (600 mg) nal trichromic Gutiérrez´VOF technique, which is a completely in freshly boiled distilled water (100 one-step tretrachrome stain (VOF Type III-G.S) ml), and let the solution cool down to room tem- composed of Light Green SF/or Fast Green FCF, perature. Methyl Blue, Orange G and Acid Fuchsin.This stain 2. Add phosphotungstic acid (1.5 g); to dissolve was applied to histological sections of whole fish it, add glacial acetic acid (3 mL) and absolute larvae and to sections of normal