Cryofixation processing is an excellent method to improve the retention of adrenomedullin antigenicity.

We reappraised the precise immunohistochemical localization of adrenomedullin (AM) by means of the combined use of the catalyzed signal amplification (CSA) system and plunge freezing (PF)/freeze substitution (FS) for light microscopy or high-pressure freezing (HPF)/FS for electron microscopy, focusing on the rat adrenal gland and heart. In the case of adrenal glands, the PF processing showed that almost all medullary cells were intensively immunoreactive, while the cortical cells showed weak immunoreaction. In the heart, almost all cardiac muscle cells of the atria were also vividly stained with the PF/FS and the CSA enhancement. On the contrary, traces of immunoreactions were seen in most of the ventricular cells. These results are consistent with the previous reports of AM radioimmunoassays and the expression of AM mRNA. However, the chemical fixation processing revealed heterogeneous immunostaining in the atrial and ventricular myocardium as well as the adrenal medulla. Intensity of the immunostaining in the chemically fixed tissues was not likely to correspond with that of AM radioimmunoassays. The HPF/FS processing clearly demonstrated the immunogold labeling on secretory granules of adrenal medullary cells as well as cardiac muscle cells of the right auricles. Immunogold labeling intensity of the cryofixed specimens was 3- to 25-fold higher than that of the chemically fixed ones.