Application of Metal Labeled Antibody Method and a Consideration of the Fixatives Suitable for Immunohistochemical Study

Abstract

1) The preparation of metal labeling antibody;Since Singer (1959) introduced the immunoferritin method, many metals have been used for labeling antibody. Metal labeled antibody has an advantage over other labeled antibodies both in its smaller molecular weight and in producing a sharp image under the electron microscope at high magnification. However, the number of metal atoms capable of conjugating with the antibody without losing antibody reactivity is quite limited, and this restriction leads to the production of insufficient electron opacity. In order to both minimize the loss of antibody titer caused by the direct labeling of heavy metal to antibody and to label as many metal atoms as possible to the antibody molecule to obtain high contrast, an indirect label of 1-(chloromercuri) ferrocene (CFM) to antibody was introduced, with thiolated egg albumin (EA) employed as an intermediate material. The method was reported in detail elsewhere (Yasuda and Yamamoto, 1975). Fig. 1 shows the location of pancreatic amylase in the zymogen granule of the acinal cell of mouse pancreas stained by CMF-labeled antibody. Many tiny grains of almost uniform size representing the location of amylase are recognized in the zymogen granules, while the grains were scarcely observed in the area outside of the zymogen granules. The concentration of the grains within the zymogen granule demonstrated by this method was heavier than that demonstrated by use of the ferritin-labelled antibody. This difference may be due to the difference in the molecular weight of the labelling materials ; The molecular weight of CMF-EA (MW. of CMF; 421; EA MW. of 45,000) is much smaller than that of ferritin (MW. 650,000 on the average). Judging from this result, this new labelling method would seem to be useful for immunohistochemical study, especially for the observation of the localization of antigens at high magnification in electron microscopy. 2) Preparation of specimens for immunohistochemistry;It is a question whether or not protein molecules such as y-globulin or its Fab fragments labeled by any kind of substance can penetrate to the cell membrane of fixed cells. As a test examination conducted in order to determine the ratio of protein penetration into the tissue block, horseradish peroxidase (MW. 39,000) was chosen as the test material in this study. Mouse liver was cut into small blocks of approximately 1 mm2 X 5 mm in size and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 3 hr, and then washed overnight with the same buffer containing 5 % sucrose. Non-frozen sections, about 40-50 , i in thickness, were prepared with a Smith-Farquhar tissue sectioner (Iran Sorvall). The

New Embedding Method for Immunohistochemical Studies Using Acrylamide Gel

“…In the immunohistochemical study on the electron microscopic level, it is desirable for the labeled antibody to react directly upon tissue antigen on the cut-face of ultrathin section, because the labeled antibody can not penetrate evenly into the fixed tissue (19). The ultrathin frozen sections (2,7,12,13,18) and the BSAembedding method (6,10) are fit for occasional purposes, but these methods are not always generalized, because the ultrastructure of the cell and tissue is not well preserved by the former method and the preparation of ultrathin sections is quite difficult in both methods.…”

“…For immunohistochemical study on the electron microscopic level it is desirable that the labeled antibody reacts directly with the antigens on the cut-face of the ultrathin sections, because the labeled antibody cannot penetrate evenly into the fixed tissue block (19). Except in special cases (5), the conventional embedding materials for electron microscopic study, such as epoxy resin and methacrylate resin, cannot be applied to immunohistochemical study for the following two reasons: 1) Most of the antigens lose their antigenicities during the embedding process of tissues, 2) The removal of embedding resin from the sections is not easy.…”