Picric acid mediated hemozoin clearance enhances immunohistochemical assessment of cytokines in pigmented rodent malarial tissues

The elimination of obscuring pigments and (or) artefacts that interfere with accurate interpretation of immunohistochemistry results is crucial towards improving specificity of antibody binding, ensuring clear results that are reliable and reproducible while forestalling false-positive or false-negative results. Extensive deposits of hemozoin pigment in tissues from malaria infected subjects constitute a hinderance towards accurate assessment of tissue and cellular antigen conformations in immunohistochemistry. Consequently, the present study aimed to establish a rapid and effective hemozoin depigmentation method for formalin fixed paraffin embedded (FFPE) mouse malarial tissues subsequent to immunohistochemistry. FFPE spleens from malarial mice with severe infections (≥ 42%) due to Plasmodium berghei ANKA parasite were subjected to depigmentation employing three (3) methods. The first and second method involved tissue incubation in 1% and 10% aqueous ammonium hydroxide solutions respectively while the third method employed saturated ethanolic picric acid solution. Optimal hemozoin pigment removal without distortion of tissue architecture or loss of antigenic epitopes was achieved upon incubation in saturated ethanolic picric acid at room temperature (26 °C) for 15 min. In contrast, both solutions of ammonium hydroxide utilized demonstrated unsatisfactory pigment removal, tissue deterioration and frequent instances of dislodged tissue(s). Saturated ethanolic picric acid solution ensured rapid and efficient depigmentation of hemozoin from tissues of malaria infected mice while preserving tissue morphology and antigenicity. Saturated ethanolic picric acid solution thus represents an effective approach for the clearance of hemozoin pigment from malarial tissue sections without compromising tissue integrity or antigenicity in immunohistochemistry.