Dako aqueous medium

After paraffin removal and rehydration, sections were either stained with hematoxylin and eosin for histological analysis or treated with 0.3% H₂O₂ for immunohistochemistry. Slides were processed as described [22 (link)] for antigen retrieval, permeabilization, blocking, and antibody staining (listed in Supplementary Table S1). For both histology and immunohistochemistry, 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).

Mice were sacrificed by cervical dislocation under anesthesia by 150 µL of a xylazine (20 mg/kg)/ketamine (200 mg/kg) solution and after cardiac puncture. Thyroid lobes were excised and either snap-frozen for RNA analysis or cut in 1 mm³ pieces for tissue dissociation and EV isolation. For histological and immunohistochemical analyses, thyroid lobes were extracted together with trachea, fixed in 4% paraformaldehyde and embedded in paraffin using a Tissue-Tek VIP-6 (Sakura, Torrance, CA, USA). Sections of 6 µm were obtained with the microtome Micron HM355S (ThermoScientific, Waltham, MA, USA). After paraffin removal and rehydration, histological sections were stained with hematoxylin and eosin, 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), as described in [28 (link)].

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.