2.14. Immunohistochemistry
Paraffin sections of thyroid samples were deparaffinized and rehydrated. Antigens were retrieved by incubating the sections in a microwave oven in sodium citrate buffer (10 mM; pH 6.0) for 15 min. Sections were brought to room temperature and rinsed with PBS. Then the sections were blocked using 5% bovine serum albumin at room temperature for 20 min. Subsequently, the tissues were incubated with primary antibodies overnight at 4˚C. Antibodies against THRβ-1 (cat. no. PA1213A), caspase-3 (cat. no. BS-0081R) and NOS-2 (cat no. sc-7271) were diluted at 1:20, 1:50 and 1:50 in PBS, respectively. After 12 h, the sections were washed with PBS and incubated for 60 min with biotin labeled goat anti-rabbit IgG secondary antibodies (cat. no. HPO3 provided by science emporium) at room temperature. Sections were stained using diaminobenzidine (DAB) chromogenic agent at room temperature for 5 min. Sections were counterstained with Mayer’s hematoxylin at room temperature for 5 min.26 The photographs were taken using a compound microscope. Finally, the mean optical density values were analyzed with ImageJ software.
2.15. Immunofluorescence
THRβ-1, caspase-3 and NOS-2 immunoreactivity was detected in the thyroid by immunofluorescence by using method of Niranjan & Srivastava27, with slight modifications. Paraffin sections of thyroid samples were deparaffinized and rehydrated. Antigens were retrieved by incubating the sections in a microwave oven in sodium citrate buffer (10 mM; pH 6.0) for 15 min. Sections were brought to room temperature and rinsed with PBS. Sections were incubated with primary antibodies overnight at 4˚C. Antibodies against THRβ-1 (cat. no. PA1213A), caspase-3 (cat. no. BS-0081R) and NOS-2 (cat no. sc-7271) were diluted at 1:20, 1:50 and 1:50 in PBS, respectively. Slides were incubated with the secondary antibody FITC at a dilution of 1:20 for 60 min at 4˚C. The sections were counterstained using DAPI for 4 minutes at RT, then slides were mounted by glycerine-based media. The immunofluorescence images were taken using EVOS 5000 Invitrogen fluorescent microscope. For semiquantitative analysis of THRβ-1, caspase-3 and NOS-2, the immunoreactive cells were analysed with Image J software.