Immunohistochemical Demonstration of the Skin Basement Membrane Antigens by the AMex ( Acetone , Methyl Benzoate and Xylene ) Method

Hur, Won; Chung, Joon; Ahn, Sung Ku; Bang, Dong Sik; Kim, Soo Chan

Abstract

BACKGROUND: Preservation of antigen determinants while retaining morphological detail is prerequisite for high quality immunohistochemistry. Conventional formalin fixation and paraffin embedding procedures are useful in preserving tissue architecture and cytologic detail. However, they destroy the antigenicity of many proteins is tissue samples. On the other hand, fresh frozen section preserve the antigenicity of most proteins, but vield poor morphological preservation.
OBJECTIVE
The purpose of this study is to evaluate the AMeX method as to the ability to preserve both antigenicity and morphologic details of the skin basement membrane zone so that precise localization of antigens can be attained in immunohistochemistry.
METHODS
Tissues were fixed in acetone at -20degrees C over night, then cleared in methyl benzoate and xylene, consecutively, and embedded in ordinary paraffin at 58-60degrees C. Sections made from this paraffinembedded tissue were stained with hematoxylin and eosin for a morphologic study and immunolabelled with antibodies against major basement membrane antigens to evaluate antigenic preservation. The staining intensity and preservation of the morphology by the AMeX method were compared with conventional formalin processed tissues and frozen tissues.
RESULTS
Morphological preservation of the AMeX method-processed sections was good throughout the epidermis, basement membrane, and dermis, and as good as that of routinely formalin-fixed paraffin-embedded sections. Frozen sections usually revealed revealed various degrees of damage by ice crystal formation throughout the epidermis to the dermis. The AMeX method-processed sections showed better or same antigenic preservation comparing the frozen sections when the sections were immunolabelled with specific monoclonal antibodies. But, when the sections were immunolabelled with patient's sera, the AMex method showed less antigenic preservation than the frozed sections. The anti-type IV collagen monoclonal antibody exhibited immunoreactivity only conventional formalin-fixed paraffin-embedded skin sections, but the intensity of the staining was weaker than the AMeX processed sections and the frozen sections.
CONCLUSION
The AMeX method can be utilized for the demonstration of skin basement membrane antigens and is superior to the fresh-frozen method in that the histologic figures are more distinct and antigencity can be preserved for a long time.