The adsorption staining technique applied to isolated premessenger ribonucleoprotein particles: a comparison with conventional techniques using electron microscope tomography.
A specific type of premessenger RNP particle, Balbiani ring granules from the dipteran Chironomus tentans, was biochemically isolated and visualized in three dimensions with electron microscope tomography. The particles were prepared for electron microscopy in three different ways: positively stained, negatively stained and adsorption-stained (embedded in polyvinyl alcohol, PVA, and concomitantly stained). The results were compared with those obtained for RNP particles studied in situ in ultrathin sections of plastic-embedded cells. The positively stained particles were compacted and heavily deformed with little or no internal structure. The negatively stained and the adsorption-stained particles were well preserved; the outer contours and the central cavities of the particles were outlined. The internal structure, i.e. the folded 7-nm elementary fibre, could not be recognized in the negatively stained particles. In the adsorption-stained particles, however, the fibre was discernable, although not quite as distinctly demarcated as in the plastic-embedded samples. We conclude that embedding in PVA with concomitant staining with uranyl acetate is a rapid method to obtain both good preservation and staining of isolated RNP particles. The PVA-embedded particles were also found to be sufficiently resistant to irradiation to permit a comprehensive tilt-series to be taken for electron microscope tomography.
Lönnroth
A
A
Department of Molecular Genetics, Medical Nobel Institute, Karolinska Institutet, Stockholm, Sweden.
Skoglund
U
U
Tokuyasu
K T
KT
Daneholt
B
B
D000327:Adsorption; D000818:Animals; D002683:Chironomidae / Q000737:chemistry / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D008854:Microscopy, Electron; D016613:Plastic Embedding; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods*; D014054:Tomography
D012261:Ribonucleoproteins