Immunohistochemical Analysis of TLR2 and TLR4 in Acanthamoeba Infection

The paraffin-embedded sections (3–5 μm) of kidneys and heart of control mice and hosts infected with Acanthamoeba spp. were stained to visualize TLR2 and TLR4 proteins. Immunohistochemistry was performed using specific primary rabbit polyclonal antibodies against TLR2 and TLR4 (Cat# sc-10739 and sc-30002, respectively; Santa Cruz Biotechnology, Inc., Oregon, USA) at a 1:500 dilution. The procedure of immunohistochemical staining is described by Kot et al. [11 (link)]. Briefly, the sections were microwaved to recover antigenicity and then incubated with primary antibodies overnight at 4 °C. Subsequently, the sections were stained with an avidin-biotin-peroxidase system with diaminobenzidine as the chromogen (Cat# K0679; DakoCytomation Inc., Carpinteria, CA, USA). The sections were washed in distilled H₂O and counterstained with hematoxylin. In negative controls, samples were not incubated with primary antibodies. Positive samples were determined microscopically by identifying brown pigmentation. Samples were evaluated using a light microscope (DM5000B; Leica, Wetzlar, Germany).