-36- 

 Immunof luorescence Microscopy 



Non-fixed ORSV-inf ected Cattleya , CMV-infected 

 Phalaenopsis and fixed (5% glutaraldehyde ) CyMV-inf ected 

 Cymbidium tissues were used. The CMV antiserum used was 

 obtained from D. E. Purcifull (Kuwite and Purcifull, 1982) 

 and the antiserum against ORSV and CyMV from G. C. Wisler 

 (Wisler et al . , 1982). The immunoglobulins ( IgG ) of CMV, 

 CyMV and ORSV antisera were purified by the use of protein 

 A-Sepharose CL-4B (Pharmacia Fine Chemicals, Sweden) as 

 described by Miller and Stone (1978). IgG was conjugated 

 with TRITC by dialysis (Hiebert et al . , 1984). Healthy 

 tissue extracts were made by triturating tissues with 10 

 volumes (w/v) of 20 mM sodium phosphate buffered saline at 

 pH 7.4 (PBS). The extracts were then filtered using 

 Whatman No. 1 filter paper. The staining solution was 

 made by mixing 27 Ail TRITC-con jugated antiserum, 27 All 

 healthy tissue extract, and 6 Ail dimethylsulphoxide (DMSO) 

 in saline (Herbert et al_. , 1982). This solution was 

 incubated for 30 minutes before tissue staining. Tissue 

 sections prepared as described in Chapter 2 were floated on 

 the above solution for another 30 minutes. After incubation, 

 tissue sections were blotted with filter paper, washed 

 three times with 20 mM PBS and incubated for another 15 

 minutes on top of a large drop (about 200 Ail) of PBS. All 

 the procedures were carried out in a moist chamber at room 

 temperature (ca. 25 C). Tissue sections were picked up 



