Heat-induced Antigen Retrieval in Conventionally Processed Epon-embedded Specimens: Procedures and Mechanisms.
We studied the effectiveness of heat-induced antigen retrieval (HIAR) in conventionally processed, epon-embedded specimens and the mechanisms of HIAR in the specimens. Frozen sections were first immunostained to examine the possibility of using HIAR for 18 antigens to avoid the effects of epoxy resin embedment. The antigenicity of 7 out of 18 antigens was retrieved with glutaraldehyde fixation followed by osmium tetroxide treatment whereas none were retrieved with glutaraldehyde fixation without post-osmication. Six antigens also exhibited positive immunostaining in semi-thin epon sections when the sections were deplasticized with sodium ethoxide followed by autoclaving. In the immunoelectron microscopy with the post-embedding method, positive reactions with fine ultrastructures were obtained using HIAR without deplasticization. These results suggested that osmium tetroxide binds to ethylene double bonds (which are introduced into protein crosslinks by glutaraldehyde) and forms an extremely stable resonance interaction with the Schiff bases, thus destabilizing the protein crosslinks. Heating also further degrades these crosslinks. The present study demonstrated that archival epon blocks can be useful resources for immunohistochemical studies for both light and electron microscopy.
Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan (SY, YO) shuji@z5.keio.jp.
Shuji
S
Yamashita
Department of Pathology, School of Medicine, Keio University, Shinjuku-ku, Tokyo, Japan (SY, YO).
Yasunori
Y
Okada
D000818:Animals; D000941:Antigens / Q000032:analysis*; D003432:Cross-Linking Reagents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004853:Epoxy Resins; D005404:Fixatives; D005976:Glutaral / Q000737:chemistry; D006358:Hot Temperature; D007150:Immunohistochemistry / Q000379:methods*; D008813:Mice, Inbred ICR; D016253:Microscopy, Immunoelectron; D009993:Osmium Tetroxide; D010047:Ovalbumin / Q000032:analysis / Q000737:chemistry; D016613:Plastic Embedding; D017208:Rats, Wistar; D012259:Ribonuclease, Pancreatic / Q000032:analysis / Q000737:chemistry
D000941:Antigens; D003432:Cross-Linking Reagents; D004853:Epoxy Resins; D005404:Fixatives; C004875:EPON; D010047:Ovalbumin; D012259:Ribonuclease, Pancreatic; D009993:Osmium Tetroxide; D005976:Glutaral
heat-induced antigen retrieval; immunoelectron microscopy; mechanism of antigen retrieval; osmicated and epon-embedded specimen