The application of cryo-ultramicrotomy and freeze-substitution in immuno-gold labelling of hybrid proteins in Escherichia coli. A comparison.

Abstract

To study the possible effects of chemical fixation upon antigenicity and structural preservation, the subcellular localization of LamB-LacZ hybrid proteins in Escherichia coli K-12 strains pop3234 and pop3299 was investigated both by cryo-ultramicrotomy and freeze-substitution. Immuno-gold labelling of sections of freeze-substituted bacteria showed the same localization of the hybrid protein as found after cryo-ultramicrotomy. The efficiency of labelling of the accumulated form of the hybrid protein was lower after freeze-substitution whereas the efficiency of labelling of the membrane-bound form showed no difference. Different fixatives and Lowicryl resins had no clear effect on the label-efficiency but the complex substitution medium, containing osmium tetroxide, uranyl acetate and glutaraldehyde, in combination with the apolar Lowicryl HM20 gave the best sectioning properties and membrane contrast. For this specific problem, although the somewhat better preservation after freeze-substitution, cryo-ultramicrotomy is to be favored since it is much less time-consuming, there are no freezing problems, ultrastructural preservation is sufficient and the theoretical benefits of freeze-substitution are not expressed.