Immunohistochemical Staining of Calcitonin and Ki-67

Immunohistochemical staining was performed for calcitonin and Ki-67, as described previously^{21 (link)}. Anti-calcitonin polyclonal (Nichirei Bioscience, Tokyo, Japan) and anti-Ki-67Monoclonal (MIB-5, Agilent, Santa Clara, CA, USA) antibodies diluted 1:50 in Dako Real™ antibody diluent (Agilent) were used. Simple Stain Rat Max-PO ® (Nichirei) or LSAB^®2 system-HRP (Agilent) were used for calcitonin or Ki-67 staining, respectively, according to the manufacturer’s instructions. Ki-67-positive cells were counted in at least five fields per rat from four to six rats for each data point at 1, 6, and 12 months after irradiation. Images were captured using a Nikon Camera Control Unit DS-L2 (Nikon, Tokyo, Japan) (400× magnification). The Ki-67-positive cells in the normal and tumor regions (Ki-67 index) were counted under the same conditions 18 months after irradiation.