Immunohistochemistry Protocol for Cell Marker Analysis

Immunohistochemistry was performed according to the method described by Camargo et al. [35 (link)]. The antibodies used for the markers and dilutions in this study are listed in Table 3. Digestion of the antigen at high temperature was performed in a pressure cooker for 1 min using citrate buffer (6.0 pH). For incubation with the primary antibody, the sections were diluted in bovine serum albumin (BSA) solution and applied to each slide. Then, the slides were incubated overnight in a humid chamber at 4 °C for 18–22 h. The slides were then washed in phosphate-buffered saline (PBS) and incubated with the secondary antibody using the ABC Kit with Vectastain (Vector Laboratories, CA, USA) (Table 3). To visualize the positive cells, the slides were washed in PBS and proteins were visualized using diaminobenzidine (DAB) chromogen (70 mg DAB in 110 mL of the Tris-HCl; Sigma Chemical Co., St. Louis, MO, USA) and Harris hematoxylin (Merck, Darmstadt, Germany), and finally mounted with microscopy resin.