title: 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 importance, 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 pressure, two immersions in xylene for 30 min each, and one in xylene plus paraffin for 45 min increased tissue preservation. An immunohistochemical 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.
authors.affiliation: Department of Animal Biology, IB, UNICAMP, Distrito de Barão Geraldo, Campinas, São Paulo, CEP 13083-970, Brazil.
authors.forename: Carina M
authors.initials: CM
authors.lastname: Souza
authors.forename: Carolina G P
authors.initials: CG
authors.lastname: Lima
authors.forename: Marcos J
authors.initials: MJ
authors.lastname: Alves
authors.forename: Wagner W
authors.initials: WW
authors.lastname: Arrais-Silva
authors.forename: Selma
authors.initials: S
authors.lastname: Giorgio
authors.forename: Arício X
authors.initials: AX
authors.lastname: Linhares
authors.forename: Patricia J
authors.initials: PJ
authors.lastname: Thyssen
mesh_terms: D019342:Acetic Acid; D000818:Animals; D002725:Chloroform; D003042:Cocaine / Q000032:analysis*; D004175:Diptera / Q000737:chemistry* / Q000502:physiology; D004772:Entomology; D000431:Ethanol; D005247:Feeding Behavior; D005404:Fixatives; D049429:Forensic Pathology; D053593:Forensic Toxicology; D005557:Formaldehyde; D007150:Immunohistochemistry; D007814:Larva / Q000737:chemistry; D009294:Narcotics / Q000032:analysis*; D010232:Paraffin; D011108:Polymers; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors; D014992:Xylenes
chemical_list: C033474:Carnoy's solution; D005404:Fixatives; D009294:Narcotics; D011108:Polymers; D014992:Xylenes; D005557:Formaldehyde; D000431:Ethanol; D002725:Chloroform; D010232:Paraffin; D003042:Cocaine; D019342:Acetic Acid; C003043:paraform
keywords: entomotoxicology; fixatives; forensic entomology; forensic science; histological technique; immunohistochemical analyses