Fluorescein isothiocyanate fitc conjugated secondary antibody

Anti-TSH receptor (TSHR), anti-nSMase, and fluorescein isothiocyanate- (FITC-) conjugated secondary antibody were obtained from Santa Cruz Biotechnology Inc. (California, USA). TSH, thyroglobulin, and Gal-3 antibodies were from Leica Biosystems (Newcastle Ltd., UK).

Anti- Signal transducer and activator of transcription-3 (STAT3), anti-TSHR, anti-Caveolin 1, fluorescein isothiocyanate (FITC)-conjugated secondary antibody were obtained from Santa Cruz Biotechnology, Inc. (California, USA); SDS-PAGE molecular weight standards were purchased from Bio-Rad Laboratories (Hercules, CA, USA). Chemiluminescence kits was purchased from Amersham (Rainham, Essex, UK). Cholesterol (CHO), TSH and cAMP EIA kits were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) and CABRU SAS (Milan, Italy), respectively. Sphingomyelin (SM) 18∶1 12∶0, SM 18∶1 16∶0, SM 18∶1 18∶1, SM 24∶0, phosphatidylcholine (PC) 16∶0 18∶1, PC 16∶0 20∶4, PC 18∶1 18∶0, ceramide 18∶1 16∶0, ceramide 18∶1 18∶0, ceramide 18∶1 20∶0, ceramide 18∶1 24∶0 were purchased from Avanti (Avanti Polar, Alabaster, USA).

Brain tissue slides for immunofluorescence staining were prepared and rehydrated in the same manner as mentioned in hematoxylin and eosin staining. The deparaffinized slides were washed twice with PBS for 10 min, treated with proteinase K at room temperature for 5 min, and then washed twice again with PBS for 10 min. To prevent non-specific reaction, tissue slices were incubated with 5% normal goat serum for 1 h at room temperature. They were continuously incubated with anti-NeuN and anti-GFAP (diluted 1:100, Santa Cruz Biotechnology) overnight at 4 °C. The slides were washed twice with PBS for 10 min and treated with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (dilution 1:100, Santa Cruz Biotechnology) for 90 min at room temperature. A drop of fluorescent mounting medium (Dako North America, Inc., Carpinteria, CA, USA) was dropped on a stained tissue and covered with cover glass. A fluorescence microscope (AXIO, Carl Zeiss Corporation, Thornwood, NY, USA) was used to observe NeuN and GFAP positive reactions. Images were captured from the cerebral cortex and selected in four random square areas (1 × 1 mm). The integrated density of NeuN and GFAP immunoreactivity was measured using Image J program (National Institute of Health, Bethesda, MD, USA).