Sc 10739

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).

Cells were homogenized in radioimmunoprecipitation assay buffer supplemented with protease and phosphatase inhibitors and centrifuged at 15,000 g for 15 min at 4°C. The protein concentration was determined according to the manufacturer’s instructions of the Pierce BCA Protein Assay kit (Rockford, IL, United States). Western blots were performed with 20–50 μg of protein. Proteins were separated in 8–10% SDS-PAGE and transferred to a nitrocellulose membrane. Western blot analysis for TLR2 (SC10739, Santa Cruz, United Kingdom, diluted 1:500), TLR4 (SC30002, Santa Cruz, United Kingdom, diluted 1:1,000), NFκB (ab16502, Abcam, United Kingdom, diluted 1:1,000), p-p65 (ab86299, Abcam, United Kingdom, diluted 1:1,000), PCK2 (ab70359, Abcam, United Kingdom, diluted 1:1,000), β-actin (AP0060, Bioworld Technology, United States, diluted 1:10,000), AKT (BS1007, Bioworld Technology, United States, diluted 1:1,000), mitochondrial transcription factor A (TFAM) (BS7319, Bioworld Technology, United States, diluted 1:1,000), COX Ⅳ (AP0707, Bioworld Technology, United States, diluted 1:1,000) were carried out according to the recommended protocols provided by the manufacturers. Images were captured by Versa Doc 4000MP system (Bio-Rad, United States) and the band density was analyzed with Quantity One software (Bio-Rad, United States).