D 6 sc13133

Histologic sections (5 μm) were obtained from 10% formalin-fixed paraffin-embedded (FFPE) blocks and stained with hematoxylin and eosin (H&E) for microscopic evaluation. Immunohistochemistry using a mouse anti-VDR monoclonal antibody (D-6: sc13133 Santacruz Biotechnology, Dallas, Texas, USA) and anti-FOXP3 (NB600–242 Novus biologicals, CO, USA) was performed on serial 5 μm -thick FFPE tissue sections on autostainer (Ventana Medical System, Tuscon, AZ) as per the manufacturer’s instructions. Nuclear and/or cytoplasmic (VDR) and nuclear (FOXP3) immunoreactivity was assessed in all cases. Scoring was done based on percentage of positive cells 0 (no staining), 1+ (< 10%), 2+ (10–50%), 3+ (> 50%) cells and semiquantitative data (0–3+) was used for correlation analysis. Cases were categorized as either focal/absent (0/1+) or diffuse (2+/3+).

Rats were euthanized by CO₂ asphyxiation. The small intestine and colon were removed and washed with ice-cold sterile PBS plus 1% (vol/vol) HALT protease inhibitor (Thermo Fisher Scientific). Samples were snap-frozen in liquid nitrogen and preserved at −80°C until protein extraction. Samples were thawed on ice and 50 mg added to 1 ml TOTEX lysis buffer, containing 1% (vol/vol) 0.1 M DTT and 1% HALT (O'Connor et al., 2004 (link)). Samples were sonicated for 5 s intervals until homogenized, then incubated for 1 h at 4°C. Protein concentration was determined using the Bradford protein assay (Bio-Rad). 30 μg of total protein from tissue lysate was added with equal volume 2xSDS to a 10% polyacrylamide gel electrophoresis system, then transferred to a nitrocellulose membrane (GE Healthcare). The membrane was then washed in PBST, blocked with 5% BSA in PBST, and incubated overnight at room temperature with mouse monoclonal VDR antibody (D-6, sc-13133, Santa Cruz Biotechnology) diluted 1:1000 in PBST. After washing, the membrane was incubated for an additional 2 h in ECL anti-mouse IgG horseradish peroxidase linked whole antibody (GE Healthcare) diluted 1:2500 in PBST. Following successive washes, the membrane was analyzed by enhanced chemiluminescence detection using a Curix 60CP film processor, as described by the manufacturer (GE Healthcare).

The UALCAN database (http://ualcan.path.uab.edu/) was used to examine the expression levels of VDR according to different histology in thyroid carcinoma. The expression of VDR in PTC cell lines and tissues was further verified by Western blot analysis. Detailed procedures of Western blot analysis were performed as described previously [41 (link)]. The antibodies used were shown as follows: antibodies against VDR (D-6, sc-13133, Santa Cruz Biotechnology, 1:500), GAPDH (10494-1-AP, Proteintech, 1:3000), HRP-conjugated affinipure goat anti-mouse IgG(H+L) (SA00001-1, Proteintech, 1:5000), HRP-conjugated affinipure goat anti-rabbit IgG(H+L) (SA00001-2, Proteintech, 1:5000).