Immunohistochemical Analysis of Tumor Protein Expression

Tumors were fixed in 4% paraformaldehyde overnight at 4°C. Then tumors were embedded in paraffin and sectioned at 5 μm. Immunohistochemistry was performed following standard procedures. After incubated with primary antibodies (Rabbit anti-GR, CST, #12041; mouse anti-NR2F1 R&D, PP-H8132-00), VECTASTAIN® ABC HRP Kit (Peroxidase, Rabbit IgG) and HRP conjugated Goat anti-mouse IgG were used, followed by ImmPACT® DAB Peroxidase (HRP) Substrate. Images were acquired on ECHO revolve microscope. Representative images of four tumors of each group were used to quantify the IHC signals of GR and NR2F1, using imageJ and the IHC Profiler plugin (Varghese et al., 2014 (link)).