Antigen-Antibodies 331 



Coons' Fluorescent Technic 



If antigens can be labeled, sources of antibody production in tissues 

 can be seen microscopically. Coons and his associates have coupled anti- 

 bodies with fluorescein isocyanate and isothiocyanate to form fluores- 

 cent carbamide-proteins. The conjugates thus formed from dye and 

 antibodies still retain the specific reaction of the antibody for the 

 antigen that causes its formation. Labeled antibodies are poured over 

 the tissues or cells and will leave a labeled protein in the tissues which 

 can be observed under a fluorescent-adapted microscope. 



Some of the solutions, antisera (immune serum), and conjugates have 

 to be prepared in the laboratory; some can be purchased,^ but it is 

 recommended that all conjugates be purified by absorption against 

 tissue powders. For reasons and means of doing this, see Coons et al. 

 {1955); Coons {1958); and Mellors {1959). The method is complicated 

 and should not be undertaken without a thorough study of the source 

 material. 



With a few exceptions (some polysaccharides) chemical fixatives must 

 be avoided to retain specific activity of the antigen. Smears, touch 

 preparations, tissue cultures, and cell suspensions can be uSed. If sec- 

 tions are preferred, freeze-dried or imfixed frozen sections cut in a 

 cryostat (page 337) are mandatory. Tissues may be quick-frozen in 

 petroleum ether previously chilled to — 65°C with dry ice-alcohol mix- 

 ture in a Dewar flask. When completely frozen, blot and store in 

 tightly stoppered test tube at — 20°C to — 70°C imtil used. Long storage 

 is to be avoided; the tissues dehydrate {Tobie, 1958). Coons et al. {1955) 

 place small pieces of tissue against the wall of a small test tube and 

 plunge it into alcohol cooled to — 70°C with solid carbon dioxide. Store 

 at — 20°C until used. 



After sectioning by either method, the tissue sections must be fixed 

 before applying the antigen-antibody solution. Fixation depends on the 

 antigen. 



fixation: proteins: 95% ethyl alcohol, sometimes absolute methyl alco- 

 hol; polysaccharides: can be fixed in the block by picric acid-alcohol- 

 formalin (Rossman fluid, page 20) and paraffin-embedded; lipids: 

 10% formalin; viruses: acetone. 



DIRECT METHOD (staiuiiig directly with fluorescein-labeled antibody 

 against specific antigen). 



Slides are dried after fixation, rinsed in buffered saline (0.8% sodium 



^ Arnel Produtts Co., \.V. (Sylvaiiia Chemicals) Baltimore Biological Laboratory, Balti- 

 more, Maryland. 



