Anti tpo

Enzyme-linked immunosorbent assay (ELISA) kits for thyroid hormone assays were purchased from Life Technologies Pvt. Ltd., India. For estimations of total cholesterol, triglyceride and high density lipoprotein cholesterol, assay kits were obtained from Span diagnostics Pvt. Ltd., Surat, India. Kits for aminotransferase enzymes, alanine transaminase, aspartate transaminase, and lactate dehydrogenase were from Erba diagnostic pvt. Ltd., GmbH, Germany. ELISA kit for TNF-α was purchased from Ray Biotech Inc, Norcross, GA, USA. While anti-TPO, anti-TSHR, anti-β-actin and nitrocellulose were obtained from Santa Cruz Biotechnology, USA; NC membrane was supplied by Millipore, USA. Jackson Immuno-Research Laboratories Inc. USA supplied HRP-conjugated anti-mouse secondary antibody. Bovine serum albumin (BSA) and Tween-20 were provided by Sigma-Aldrich, USA. All other routine chemicals used in the biochemical studies were purchased from Hi-Media, Mumbai, India.

Equal amounts of protein from different samples were subjected to 10% SDS-PAGE, followed by electrotransfer from the gel to polyvinylidene difluoride membranes (Millipore). The membranes were incubated overnight at 4°C with anti-NIS (1 : 250, Biorbyt), anti-TPO (1 : 200, Santa Cruz), anti-Tg (1 : 10000, Abcam), and anti-BiP (1 : 1000, Proteintech). GAPDH protein was evaluated as a loading control (1: 3500, Proteintech). Immune complexes were detected using the FluorChem Q Gel Imaging and Analysis System (Protein Simple, California, USA), and band densitometry was performed through the use of associated densitometry software (Protein Simple, California, USA).

Human primary thyrocytes were treated without or with 0.2 mM palmitic acid for 72 hours. The cells were washed with PBS and fixed with 4% paraformaldehyde. Then, the cells were washed and blocked with 10% goat serum and incubated with anti-NIS (1 : 100, Biorbyt), anti-TPO (1 : 100, Santa Cruz), and anti-Tg (1 : 50, Abcam) at 4°C overnight, respectively. After further washing, the thyrocytes were incubated with secondary TRITC- or FITC-conjugated goat anti-mouse IgG. Nuclei were stained with 4, 6-diamidino-2-phenylindole (DAPI; Vector Laboratories) and the resultant immunofluorescence was viewed under a fluorescent microscope (Axio Imager 2, Carl Zeiss, Jena, Germany). All images were acquired using the same exposure time. As a negative control, the identical procedure was performed in the absence of the primary antibody and replaced by PBS. The semiquantitative analysis of fluorescence intensity was conducted using Image J software.