Immunohistochemistry and Immunofluorescence Staining

After paraffin removal and endogenous peroxidases inhibition with 0.3% H₂O₂ for immunohistochemistry, the slides were treated as described in [29 (link)] for antigen retrieval, permeabilization and the blocking of non-specific sites. Anti-phospho-ERK primary antibody (Cell Signaling Technology, Leiden, The Netherlands, #4376; 1/200) was incubated overnight at 4 °C. HRP-conjugated secondary antibody was incubated in PBS/10% BSA/0.3% Triton X-100 for 1 h at room temperature (RT). Diaminobenzidine was used for the revelation of phospho-ERK immunostained cells while hematoxylin staining allowed the visualization of tissue structure. Finally, slides were mounted in Dako aqueous medium (Agilent Technologies, Santa Clara, CA, USA) and scanned with the panoramic P250 digital slide scanner (3DHistech, Budapest, Hungary). For immunohistofluorescence, thyroid lobes were embedded in gelatin and frozen sections of 6 µm were obtained with the cryostat (ThermoScientific, Waltham, MA, USA), as described in [30 (link)]. Anti-CD206 primary antibody was incubated overnight at 4 °C. Secondary antibody coupled to Alexa-488 (Invitrogen, Waltham, MA, USA) and fluorescent nuclear dye (Hoechst 33258; Merck, Kenilworth, NY, USA) were used. Slides were observed with the Zeiss Cell Observer Spinning Disk confocal microscope.