**7. Concluding remarks**

The main mechanisms of HIAR are cleavage of chemical crosslinks formed by formaldehyde and extend of polypeptides chains to expose epitopes. Highly masked epitopes in heat-stable proteins can be exposed with reduction of disulfide bond followed by heating. Heating also cleaves crosslinks formed by double fixation with glutaraldehyde and osmium tetroxide. The principle of HIAR is applicable for immunoelectron microscopy using the post-embedding and pre-embedding methods, in which tissues are fixed with a standardized fixative described in this text. The association of exposed epitopes and antibodies under a suitable solution are also important for each immunohistochemical reaction.
