Image labtm software version 5

As carried out earlier^{61 (link)}, individual protein samples (20 μg each) extracted from thyroid gland were separated on a 12% SDS-polyacrylamide gel and then transferred on to a nitrocellulose membrane electrophoretically. The membrane was blocked in a 5% BSA and 0.5% Tween-20 for 1 h with mild shaking at room temperature followed by an overnight incubation at 4 °C with the primary antibodies, anti-TPO (1:2000 dilutions), anti-TSHR (1:2000 dilutions) and internal loading control β-actin (1:2000 dilutions) overnight at 4 °C. After washing thrice with sterile phosphate buffer saline (PBS) and 0.05% Tween-20, incubated with horseradish peroxidase (HRP) -conjugated secondary anti-mouse antibody (1:5000 dilutions) for 1 h at room temperature. The membrane was then developed using enhanced chemiluminescence (ECL), Thermo Scientific, Pierce, USA, and visualized using ChemiDoc^TM MP Imaging system (Bio-Rad, USA). Western blot images were quantified using image Lab^TM software version 5.1 (Bio-Rad).

Hippocampal tissues were homogenized in a lysis buffer and the protein content was measured using a Bradford assay kit (Bio-Rad, USA). Briefly, equal amounts of protein (20 μg) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Pierce, Rockford, IL) using a Bio-Rad Trans-Blot system. The membranes were blocked with a blocking solution composed of 20 mM Tris-Cl (pH 7.5), 150 mM NaCl, 0.1% Tween 20 and 3% bovine serum albumin and incubated overnight at 4 °C with one of the following primary antibodies (1:1000): Aβ (1–42), β-secretase 1 or β-actin obtained from Thermo Fisher Scientific Inc. (Rockford, IL). The filters were washed and subsequently probed with peroxidase-labeled secondary antibodies. Finally, the band intensity was analyzed using a ChemiDoc imaging system with Image LabTM software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA). The results were presented as arbitrary units after normalization to levels of the β-actin protein expression.

After protein extraction from striatal tissues using RIBA lysis buffer (Bio Basic Inc., Markham Ontario, L3R 8T4 Canada), equal amounts of protein were separated on a poly-acryl amide gel by SDS-PAGE using a Bio-Rad Mini-Protein II system. Polyvinylidene difluoride membranes was used to transfer the protein (Pierce, Rockford, IL, USA) with a Bio-Rad Trans-Blot Turbo system. Immunodetection of Western blots was conducted by incubating the membranes at room temperature for 1 h with blocking solution composed of 20 mM Tris–Cl (pH 7.5), 150 mM NaCl, 0.1% Tween 20 and 3% bovine serum albumin. Membranes were incubated overnight at 4 °C with one of the following primary antibodies (1:1000): p-(Tyr 1022/1023) JAK1 (Cat. no. 700028), p-(Tyr 1007–1008) JAK2 (Cat. no. PA5-85,735), p-(Tyr 705) STAT3 (Cat. no. 710093) and β-actin (Cat. no. MA5-15,739) obtained from Thermo Fisher Scientific Inc. (Rockford, IL). After washing, peroxidase-labeled secondary antibodies were added, and the membranes were incubated at 37 °C for 1 h. Analysis of the band intensity was performed using ChemiDoc™ imaging system with Image LabTM software version 5.1 (Bio-Rad Laboratories Inc., Hercules, CA, USA). The results are expressed as arbitrary units after normalization to β-actin protein expression.