Immunohistochemistry and imaging of adrenal slices

All animal procedures were approved by the Massachusetts Institute of Technology (MIT) Committee on Animal Care. For c-fos expression analysis, rats were anesthetized at least 1 week after MNP injection and were stimulated with AMF for 40 s. After stimulation, rats were kept anesthetized for 60 min to allow for c-fos expression. For all immunostaining, rats received intraperitoneal injection with Fatal-Plus Solution (100 mg/kg in PBS) and were transcardially perfused with 4% paraformaldehyde (PFA; Electron Microscopy Sciences) in PBS. For examination of long-term effects and efficiency of the magnetothermal stimulation, a group of rats injected with MNPs were exposed to repeated stimulations over a period of up to 2 months, and on the last stimulation, rats were perfused as described above.

Following perfusion, the adrenal gland was extracted from the rat and fixed in 4% PFA overnight. After three washes with PBS, all adrenal glands were embedded in 5% agarose, after the agarose was solidifies the adrenal gland was sliced into 40-μm slices using a vibrating blade microtome (Leica VT1000S). Slices were then permeabilized with 0.3% (v/v) Triton X-100 and blocked with 5% goat/donkey serum in PBS for 30 min. Slices were incubated overnight at 4°C in a solution of primary antibodies and 5% goat/donkey serum (depending on secondary) in PBS. After three washes of the slices with PBS, incubation with secondary antibody in PBS was performed for 3 hours and with DAPI (4′6-diamidino-2-phenylindole) (1:50,000) for another 20 min and washed three times with PBS. Fluoromount-G (SouthernBiotech) was used for mounting slices onto glass microscope slides. A laser scanning confocal microscope (FluoView FV1000, Olympus) was used for imaging with 4×, 20×, and 60× objectives. Mosaic images of the entire slices were generated by the FV1000 software (Olympus). H&E was performed on the adrenal slices using the manufacturer’s standard protocol, and after mounting, slices were imaged using EVOS imaging system.