Standardization of Histological Procedures for the Detection of Toxic Substances by Immunohistochemistry in Dipteran Larvae of Forensic Importance

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.