Unfixed tissue for electron immunocytochemistry: a simple preparation method for colloidal gold localization of sensitive epitopes using ethanediol dehydration.
A quick, simple protocol is described for the preparation of tissue for electron immunocytochemistry without the use of fixatives or deleterious solvents. Fresh, normal human colon was rapidly dehydrated in ethanediol (ethylene glycol) then embedded directly in low-acid glycol methacrylate. Using both mono- and polyclonal antibodies, in conjunction with colloidal gold probes, a range of intra- and extracellular epitopes were localized; these epitopes included lysozyme, chromogranin, desmin and collagen IV. Overall, the tissue compared well with material fixed in glutaraldehyde, partially dehydrated and embedded in LR White acrylic resin. Ultrastructural detail was good and was further enhanced, without affecting probe density and epitope localization, by the addition of 1% tannic acid or 1% uranyl acetate to the dehydrant. The technique is applicable to a wide range of tissues, allowing excellent antigen retention which might prove useful for the immunolocalization of sensitive epitopes.
Stirling
J W
JW
Department of Pathology, Flinders Medical Centre, Bedford Park, Australia.
D000911:Antibodies, Monoclonal; D003106:Colon / Q000737:chemistry* / Q000276:immunology / Q000648:ultrastructure; D003890:Desiccation; D000939:Epitopes / Q000032:analysis*; D019855:Ethylene Glycol; D005026:Ethylene Glycols; D006046:Gold; D006801:Humans; D007150:Immunohistochemistry; D008689:Methacrylates; D016253:Microscopy, Immunoelectron / Q000379:methods*; D016610:Tissue Embedding
D000911:Antibodies, Monoclonal; D000939:Epitopes; D005026:Ethylene Glycols; D008689:Methacrylates; C005044:hydroxyethyl methacrylate; D006046:Gold; D019855:Ethylene Glycol