*2.3. Immunohistochemical (IHC) Staining*

A representative FFPE section from each tissue block (5 μm thickness) was deparaffinized, hydrated with series of graded alcohol and then antigen retrieval at 115 ◦C for 3 min was performed in Tris-EDTA buffer (10 mM Tris base, 1 mM EDTA, 0.05% Tween 20, pH 9.0) as described previously [40]. Tissues were treated with 3% H2O2 in Tris-buffered saline for 8 min to block the endogenous peroxidase activity. Then slides were incubated with a background punisher to block non-specific staining (Biocare Medical, LLC, Concord, CA, USA) for 20 min, and were followed by 1 hr incubation with rabbit anti-Gal-3 antibody at a 1:500 dilution (ab53082, Abcam, Cambridge, MA, USA). The rinsed sections were then incubated with biotinylated anti-rabbit secondary antibody for 20 min, subsequently detected using The VECTASTAIN ABC System (Vector Labs, Burlington, ON, Canada) and peroxidase substrate diaminobenzidine (DAB) as the chromogen until staining signal developed. Sections of a classical variant of PTC known with Gal-3 positivity were included in each batch of immunostaining for Gal-3 antibody incubation to serve as an external positive control as well as for an irrelevant isotype specific IgG in place of the primary antibody as a negative control to exclude a nonspecific staining. All slides were counterstained with hematoxylin and viewed under a light microscope.
