Quantitative immuno-gold labelling and ultrastructural preservation after cryofixation (combined with different freeze-substitution and embedding protocols) and after chemical fixation and cryosectioning

Summary Among the variety of parameters affecting immuno-gold labelling efficiency, mainly the effects of different preparative protocols were tested. Preservation of ultrastructure and of antigenicity are the salient features of this study. We have labelled insoluble components of the secretory matrix of Paramecium trichocysts with specific antisera, using 10 nm colloidal gold particles. The highest labelling efficiency was obtained with fast freezing (cryofixation, either by sandwich or spray-freezing), freeze-substitution in methanol (without added fixatives) and hydrophilic Lowicryls, particularly when applied at low temperatures (K11M at 193 K). The presence of different chemical fixatives always reduced the labelling density and some recommendations from the literature do not appear advisable. Methods commencing with fixation at 0° C, such as progressive lowering of temperature (PLT) or preparation of cryostat sections, i.e. with chemical pretreatments, always resulted in lower labelling density. Our data appear, therefore, relevant for optimal immuno-gold labelling of insoluble antigens and emphasize the potential of cryofixation as a primary preparation step. In addition, ultrastructural preservation was also superior after cryofixation.Abbreviations AB antibodies - AED aminoethyldextran - Au₁₀ colloidal gold (10 nm diameter) - BSA bovine serum albumin - EtOH ethanol - FA formaldehyde - GA glutaraldehyde - LN₂ liquid nitrogen - MeOH methanol - pA protein A - Pipes piperazine-N,N-bis(2-ethanesulphonic acid) - PLT progressive lowering of temperature - PVP polyvinylpyrrolidone - UA uranylacetate