Specific primary rabbit polyclonal antibodies

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).

Paraffin-embedded sections (3–5 μm) of brains from mice infected with Acanthamoeba isolated from patients and from Malta Lake (control and 2, 4, 16, 30 dpi) were immunostained for visualization of TLR2 and TLR4 proteins.
Immunohistochemistry was performed using specific primary rabbit polyclonal antibodies against TLR2 and TLR4 (Santa Cruz Biotechnology, Inc., cat. no. sc-10739 and sc-30002) in a final 1:500 dilution. Firstly, the deparaffinized sections were microwave irradiated in citrate buffer (pH 6.0) to heat induce epitope retrieval. After slow cooling to room temperature, slides were washed in PBS twice for 5 min and then incubated with primary antibodies overnight (4 °C). On the next day, sections were stained with an avidin-biotin-peroxidase system with diaminobenzidine as the chromogen (Rabbit ABC Staining System, Santa Cruz Biotechnology, Inc., cat. no. sc-2018) in conformity with staining procedure instructions included. Sections were washed in distilled H₂O and counterstained with hematoxylin. For a negative control, specimens were processed in the absence of primary antibodies. Positive staining was defined microscopically by visual identification of brown pigmentation. The IHC-stained sections were examined by light microscope (Leica, DM5000B, Germany).