Thyroid glands, lungs and inguinal fat were dissected and fixed in 10% neutral-buffered formalin (Sigma-Aldrich, St. Louis, MO, USA) and subsequently embedded in paraffin. Five-micrometer-thick sections were prepared and stained with hematoxylin and eosin. For each animal, single random sections of thyroid were examined. For thyroids, morphologic evidence of hyperplasia, capsular invasion, and vascular invasion was routinely examined in that single section.
Immunohistochemistry (IHC) was conducted as previously described with some modifications [50 (link)]. For the antigen retrieval step, slides were heated in 0.05% citraconic anhydride solution (pH 7.4; Sigma-Aldrich, St. Louis, MO, USA) at 98°C for 60 minutes followed by treatment with rabbit anti-p-STAT3 antibody (1:100 dilution, Cell Signaling, Denver, MA, USA) and anti-cleaved caspase 3 antibody (dilution 1:300, cat. 9661, Cell Signaling) at 4°C overnight. The antigen signals were detected by treatment with the peroxidase substrate diaminobenzidine, followed by counterstaining with Gill's hematoxylin (Electron Microscopy Sciences, Hatfield, PA, USA). Relative positive cell ratio was quantified by using NIH IMAGE software (Image J 1.47).
Immunohistochemistry (IHC) was conducted as previously described with some modifications [50 (link)]. For the antigen retrieval step, slides were heated in 0.05% citraconic anhydride solution (pH 7.4; Sigma-Aldrich, St. Louis, MO, USA) at 98°C for 60 minutes followed by treatment with rabbit anti-p-STAT3 antibody (1:100 dilution, Cell Signaling, Denver, MA, USA) and anti-cleaved caspase 3 antibody (dilution 1:300, cat. 9661, Cell Signaling) at 4°C overnight. The antigen signals were detected by treatment with the peroxidase substrate diaminobenzidine, followed by counterstaining with Gill's hematoxylin (Electron Microscopy Sciences, Hatfield, PA, USA). Relative positive cell ratio was quantified by using NIH IMAGE software (Image J 1.47).