Immunogold labeling of luciferase in the luminous bacterium Vibrio harveyi after fast-freeze fixation and different freeze-substitution and embedding procedures.
We studied the ultrastructural localization of luciferase on sections of the bioluminescent bacterium Vibrio harveyi by indirect immunogold staining, using a polyclonal antiluciferase antibody and the usual control tests, after chemical fixation or fast-freeze fixation (FFF) followed by different freeze-substitution (FS) procedures and embedding in either Epon or LR White. After liquid fixation with glutaraldehyde and paraformaldehyde and LR White embedding, labeling occurred over the cytoplasm but not over the condensed nucleoid. Epon embedding almost abolished it. FFF-FS considerably improved the morphological preservation and revealed cytoplasmic "patches" with a complex ultrastructure in Epon sections. The preservation was always less good in LR White. The patches were densely labeled, even in Epon sections, after FS in acetone. However, labeling intensity was 3.7 times greater in LR White than in Epon. With both resins, labeling diminished similarly when fixative agents were present in the FS medium. The localization of luciferase in the cytoplasm and particularly in the patches is discussed.
Nicolas
M T
MT
Laboratoire de Bioluminescence et Laboratoire de Technologie Appliquée à la Microscopie Electronique, Paris, France.
Bassot
J M
JM
Nicolas
G
G
D005615:Freezing; D007150:Immunohistochemistry / Q000379:methods; D008156:Luciferases / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D014733:Vibrio / Q000201:enzymology* / Q000648:ultrastructure
D008565:Membrane Proteins; D008156:Luciferases