In attempting to improve specificity of staining, the fluorescein labeled antisera used in both direct and indirect methods were treated in one of several ways: (1) They were passed through Dowex-2-chloride twice and treated with acetone insoluble powders (Coons, 1958) prepared from mouse liver or from healthy sweet clover stems or crown gall tissue produced by Agrobacterium tumefaciens (E. F. Smith + Townsend) Conn, on sweet clover stems. (2) The conjugates as well as the intermediate sera were absorbed for 30 minutes with 20 -- 50 mg of proteins extracted from healthy sweet clover stems. The proteins were extracted with 3 volumes of ** f in ** f to give a nearly neutral extract and precipitated by 80% saturation with ** f. The precipitate was washed twice with an 80% saturated solution of ** f, dissolved in a small quantity of 0.1 M neutral phosphate buffer, dialyzed against cold distilled water till free from ammonium ions, and lyophilized using liquid nitrogen. (3) In other experiments the indirect conjugate was treated with 3 volumes of ethyl acetate as recommended by Dineen and Ade (1957). (4) The conjugates were passed through a diethylaminoethyl (DEAE --) cellulose column equilibrated with neutral phosphate buffer (PBS) containing ** f potassium phosphate and ** f.

The technique of cutting sections was essentially the same as that described by Coons et al. (1951). Root and stem tumors from sweet clover plants infected with WTV were quick-frozen in liquid nitrogen, embedded in ice, and cut at 3 -- 6 | m in a cryostat maintained at -- 16 ` to -- 20 `. The sections were mounted on cold slides smeared with Haupts' adhesive (Johansen, 1940) in earlier experiments, and in later experiments with a different mixture of the same components reported by Schramm and Rottger (1959). The latter adhesive was found to be much more satisfactory. The sections were then thawed by placing a finger under the slide and dried under a fan for 30 minutes; until used they were stored for as long as 2 weeks.