APPLICATION OF METAL LABELED ANTIBODY METHOD AND A CONSIDERATION OF THE FIXATIVES SUITABLE FOR IMMUNOHISTOCHEMICAL STUDY

NOBORU YAMAMOTO
1977 Acta Histochemica et Cytochemica  
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
more » ... his 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
doi:10.1267/ahc.10.246 fatcat:ieiyhxjvnffpppfi7v7ggdbuze