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.