Results of specific staining by the direct and the indirect methods were similar and showed the localization of WTV antigen in certain tissues of tumors. The virus antigen was concentrated in the pseudophloem tissue. Frequently a few isolated thick walled cells or, rarely, groups of such cells in the xylem region, were also specifically stained, but there was no such staining in epidermis, cortex, most xylem cells, ray cells, or pith.

Within the pseudophloem cells the distribution of WTV antigen was irregular in the cytoplasm. No antigen was detectable in certain dark spherical areas in most cells. These areas are thought to represent the nuclei. In some tumor sections small spherical bodies, possibly inclusion bodies (Littau and Black, 1952) stained more intensely than the rest of cytoplasm and probably contained more antigen. In all cases studied tissues of the stem on which the tumor had developed did not contain detectable amounts of WTV antigen.

In both the direct and indirect methods of staining, the conjugates had nonspecifically staining fractions. In the indirect method, this was evident from the fact that tumor sections were stained light green even when stained with NS and ** f or with ** f only. In the direct method, ** f, not further treated, stained certain tissues of healthy sweet clover stems nonspecifically and WTV tumor sections were similarly stained by comparable ** f. After ** f and ** f were passed through Dowex-2-chloride twice and treated twice with healthy sweet clover tissue powder, nonspecific staining was greatly reduced but a disturbing amount of such staining was still present. Treatment of the conjugates with ethyl acetate, and the conjugates (which had been passed through Dowex-2-chloride) with mouse liver powder, sweet clover crown gall tissue powder, or healthy sweet clover proteins did not satisfactorily remove nonspecifically staining substances in the conjugates. Such treatments of the conjugates have usually been successful in eliminating nonspecific staining in several other systems (Coons, 1958). Schramm and Rottger (1959) did not report any such nonspecific staining of plant tissues with fluorescein isocyanate labeled antiserum to tobacco mosaic virus. The reason for the failure of these treatments to eliminate nonspecific staining in the conjugates in our system is not known.