Bleaching of Melanomacrophages from Tissues of Ectothermic Vertebrates for Later Use of Immunohistochemical and in Situ Hybridization Technique
Abstract
Due to the large quantity of melanomacrophages in the organs of the ectothermic vertebrates, with special interest in the ranids and fish, with their brownish melanin granules, we decided to test the MELANIN removal technique, in order to facilitate the observation of the organ fragments in the slides, under the direct light optical microscope, when using the antibodies and biotinylated probes.
Thus, the melanin bleaching study favored the visualization of the diaminobenzidine chromogen (DAB) without interfering with the antigen-antibody affinity of immunohistochemistry and without interfering with the technique by which specific nucleotide sequences are identified in histological sections. (of DNA or RNA, endogenous, bacterial or viral).
This bleaching of melanin from tissues avoided false positive results, without interfering with the IHQ and ISH techniques for Mycobacterium spp and Francisella spp in fish.
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Introduction
Fish, especially teleosts as well as amphibians, share with the ectothermic vertebrates the existence of an extra-cutaneous pigment system consisting of large, irregular cells in various tissues and organs that can produce and storing melanin within them. This variable amount of melanomacrophages centers or macrophage aggregates are usually found on a larger scale within the endothelial reticulum in the hematopoietic matrix, in the spleen and kidney, but also in the liver (MESSEGUER et al., 1994) and to a lesser extent in the submucosa of the intestine, thymus, gills, brain and gonads of teleosteal fish. The emergence of these structures is related to several factors and conditions, such as organ, age, nutritional status, anatomopathological conditions, as well as environmental changes such as in the processes of detoxification by pollutants and cytoprotective functions related to free radicals.
In the liver, kidneys and spleen, the MMs may contain melanin, cellular fragments, hemosiderin granules and lipofucsine residues, mainly due to their functions of cellular debris sequestration and potentially toxic materials, in addition to participating in the response of fishes and ranids to infectious agents such as fungi and bacterias (STEINEL, BOLNICK, 2017)
The IHQ technique uses the enzyme peroxidase and DAB (3'-3-diaminobenzidine tetrahydrochloride) as the most common and inexpensive chromogen and can have its reaction visualized in a direct light optic microscope, allowing, for example, the diagnosis of various agents’ infectious diseases. DAB, however, presents as a marker of deep brown color and may become indistinguishable from the brownish granules of melanin, causing diagnostic errors when viewed under the microscope.
The ISH reaction allows to accurately localizing in the paraffin or frozen tissue, a specific gene or its transcripts. The technique also allows to associate the presence of DNA or mRNA of microorganisms with the morphology, or even to relate the presence of genes and their transcripts with the pathological processes. Thus, there is the association with immunohistochemistry and the ribosondes can be used to study, for example, viral, bacterial, etc. infections, and to differentiate productive viral infections from non-productive ones.
The purpose of this work was to standardize a simple and efficient protocol to bleach the melanin in heavily pigmented tissues. The bleaching technique allowed a correct observation of the chromogen in the antibody-antigen (IHQ) reaction and in the ISH probes in the tissue, not interfering with their affinity and thus avoiding false positives.
Conclusion
Among the cutaneous pigment macromolecules, we have melanin, eumelanin and pheomelanin (BILLINGHAM & SILVERS 1960, LING 1974). The extra-cutaneous melanin found in melanomacrophages tissues of ectothermic vertebrates is formed by the conversion of tyrosine into alpha-3, 4-dihydroxyphenylalanine and then into dopaquinone and melanin by tyrosinase (WOOLF & SWAFFORD, 1988). There are numerous functions attributed to melanomacrophages such as: antioxidant function, helping to protect the lipids of cell membranes from free radical attack (STEINEL, NC, BOLNICK, 2017), phagocytosis of resistant pathogens such as spores of parasites and bacterias (ROBERTS, 1975); processing of antigens in the immune response (AGIUS, 1985); destruction, detoxification or recycling of endogenous and exogenous materials (FERGUSON, 1976; ELLIS, 1980, HERRAEZ; ZAPATA, 1986); and the response to foreign bodies, including infectious agents (AGIUS, ROBERTS, 2003). Melanomacrophage centers are associated with the presence of acid-resistant intracellular bacteria such as mycobacteria and parasites such as Myxobolus spp. (ROBERTS, 2001). There is evidence that melanomacrophages play a relevant role in the control of myxosporidia infections (SUPAMATTAYA et al., 1993).
Bleaching is by gradual oxidation by hydrogen peroxide (KORYTWSKI & SARNA, 1990) and it was evident from our work that the visualization and analysis of IHQ and ISH for francisella and mycobacterium with DAB chromogen use will not be overestimated leading to false positives. This is especially interesting when doing computerized image analysis. These same results were obtained by SILVA, A P and collaborators, in 2011, using the melanin whitening in the epidermis of the South American sea lion for later application of enzymatic immunohistochemistry. It is concluded to be an effective whitening technique for any histological or histopathological study in any animal tissue with melanin.
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