In the first few experiments ** f was passed through Dowex-2-chloride twice and absorbed twice with 50 -- 100 mg sweet clover tissue powder. The intermediate sera were also similarly absorbed with tissue powder. Sections of sweet clover stem and root tumors were treated with 1: 10 solution of ** f for 30 minutes, washed in buffered saline for 15 minutes, stained with ** f for 30 minutes, and washed for 15 minutes in PBS. Such sections showed bright yellow-green specific fluorescence in the cells of the pseudophloem tissue (Lee and Black, 1955). This specific fluorescence was readily distinguished from the light green nonspecific fluorescence in consecutive sections stained with 1: 10 dilution of NS and ** f or with ** f alone. Unstained sections mounted in buffered glycerine or sections treated only with NS or ** f did not show such green fluorescence. Sections of crown gall tissue similarly stained with either ** f and ** f or NS and ** f also showed only the light green nonspecific fluorescence. However, the nonspecific staining by the ** f in tumor sections was considered bright enough to be confused with the staining of small amounts of WTV antigen.

Two absorptions of ** f with ethyl acetate or two absorptions of ** f (which had been passed through Dowex-2-chloride), NS and ** f with crown gall tissue powder, or mouse liver powder did not further improve the specificity of staining. Treatment of all the sera with sweet clover proteins greatly reduced nonspecific fluorescence, especially when the treated conjugate was diluted to 1: 2 with 0.85% saline. In all the above procedures, when the intermediate sera were diluted to 1: 10 or 1: 100 with 0.85% saline, the specific and nonspecific fluorescence were not appreciably reduced, whereas, a dilution of the intermediate sera to 1: 500 or diluting the ** f to 1: 5 greatly reduced specific fluorescence. Rinsing the sections with PBS before layering the intermediate sera did not improve the staining reaction. In addition to other treatments, treating the sections with normal sheep serum for half an hour before layering ** f did not reduce nonspecific staining.