TECHNICAL NOTE PATHOLOGY/BIOLOGY Carina M. Souza, 1 M.Sc.; Carolina G. P. Lima, 1 M.Sc.; Marcos J. Alves-Jr, 1 B.Sc.; Wagner W. Arrais-Silva, 2 Ph.D.; Selma Giorgio, 1 Ph.D.; Arício X. Linhares, 1 Ph.D.; and Patricia J. Thyssen, 1 Ph.D. Standardization of Histological Procedures for the Detection of Toxic Substances by Immunohistochemistry in Dipteran Larvae of Forensic Importance* ABSTRACT: Immunohistochemistry (IHC) technique is an alternative toxicological analysis to detect drugs in insects of forensic impor- tance, but it requires thorough histological procedures. In this study, we tested different fixatives—phosphate-buffered paraformaldehyde 4% (PP), Carnoy’s fluid (CF), Kahle’s solution (KS), ethanol in different concentrations, and ethanol associated to PP and CF, time of fixation and histological processes for dipteran larvae’s tissue, aiming to develop a sample preparation protocol for IHC application. A suitable fixation was achieved using PP for 12 and 24 h, CF for 3 h, 70% ethanol for 19 days, and 70% ethanol/CF for 2 h/3 h. Postfixation using negative pres- sure, two immersions in xylene for 30 min each, and one in xylene plus paraffin for 45 min increased tissue preservation. An immunohisto- chemical test for cocaine detection was performed using monoclonal benzoylecgonine antibody from mouse, peroxidase-conjugated anti-mouse IgG and visualized by 3,3′-diaminobenzidine method showed these histological procedures didn’t compromise antigenicity. KEYWORDS: forensic science, forensic entomology, entomotoxicology, histological technique, immunohistochemical analyses, fixatives Necrophagous insects feed on organic remains of dead animals to obtain the protein source needed to ovarian development and to stimulate oviposition/larviposition. These larvae develop on carcasses affecting the decay process (1). Therefore, these entomo- fauna can be useful in medico-criminal investigations, litigations on human and animal pests, and stored food contamination, sub- jects approached by forensic entomology (2,3). Sarcosaprophagous entomofauna can be also an alternative source to provide information about toxicological analyses when human tissues, blood, or urine are not available due to the advanced stage of decomposition of a corpse (4). Entomotoxicol- ogy aim at detecting toxic substances in larvae tissue and inves- tigating the effect and interference caused by these drugs in the development of insects with the purpose to estimate a postmor- tem interval more accurately (5,6). Detection of chemical substances in necrophagous insects is possible because immature specimens feed on contaminated human tissues introducing into their metabolism drugs and toxins that can be accumulated allowing their identification (6). The techniques performed to insect specimens are basically the same used to human tissues and biologic fluids: gas chromatography/ mass spectrometry, liquid chromatography/mass spectrometry, thin-layer chromatography, radioimmunoassay, and immunohis- tochemistry (IHC) (7–28). Immunohistochemical technique is a promising alternative approach to drug detection in insect, because it is more specific than the traditional assays, and a great advantage of this method- ology is the information concerning the pattern of topographic distribution of drugs in larval tissues, generating pharmacody- namics and pharmacokinetics data (29). Bourel et al. (21), for example, studying morphine metabolism in Calliphora vomitoria L. (Diptera: Calliphoridae) larvae demonstrated, by IHC, the accumulation of this drug in the area between endo- and exocuti- cle. Furthermore, it is known that such substances as morphine can be rapidly and efficiently excreted by larvae, but a low concentration, usually lower than that observed in the food source, can be distributed and sequestered into different parts of the larval body, such as hemolymph, fat bodies, and/or cuticle, remaining incorporated within the larval tissues through the different stages of development (30). A fact that must be observed in preparation of entomologi- cal samples for histological studies is that substantial differ- ences related to morphological and integumental characteristics among different fly species may require a new kind of stan- dardization. The literature on this issue is rare, and there is no specific data for histology of necrophagous dipterans (31–34). Besides, choosing the correct fixative and time of fixation that 1 Department of Animal Biology, IB, UNICAMP, Distrito de Bar~ ao Geraldo, Campinas, S~ ao Paulo, CEP 13083-970, Brazil. 2 Campus of Araguaia, UFMT, Rodovia MT-100, Km 3,5, Pontal do Araguaia, Mato Grosso, CEP 78698-000, Brazil. *Supported by FAPESP (The State of S~ ao Paulo Research Foundation) through a thematic project (Grant # 2004/08544-0) and scholarship grants (Grants # 2005/54480-7 and # 2006/50564-4). Received 16 Nov. 2011; and in revised form 6 Mar. 2012; accepted 2 June 2012. © 2013 American Academy of Forensic Sciences 1015 J Forensic Sci, July 2013, Vol. 58, No. 4 doi: 10.1111/1556-4029.12140 Available online at: onlinelibrary.wiley.com