Freeze-substitution without aldehyde or osmium fixatives: ultrastructure and implications for immunocytochemistry.
Cryo-fixation followed by freeze-substitution without aldehyde or osmium fixation has been investigated as a method for preparing biological specimens with a view to minimizing antigenic alteration. Samples of both solid tissues (mouse small intestine and human kidney) and a human tumour cell line grown in vitro were rapidly frozen by impact (slammed) onto a copper block cooled with liquid nitrogen. They were freeze-substituted at -80 degrees C in methanol, and embedded at low temperature in Lowicryl K4M or HM20. Resin blocks were polymerized by ultraviolet light. Well-preserved ultrastructure was observed in the outer 10-15 microns of all samples. Positive immunocytochemical localization of fixation-resistant and fixation-labile antigens was obtained on sections of human kidney and the human breast tumour cell line ZR-75-1 at both light and electron microscope levels.
Institute of Cancer Research, Haddow Laboratories, Sutton, Surrey.
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Monaghan
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Robertson
D000818:Animals; D001940:Breast / Q000276:immunology / Q000648:ultrastructure; D003094:Collagen / Q000276:immunology; D015925:Cryopreservation; D005404:Fixatives; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D007421:Intestine, Small / Q000276:immunology / Q000648:ultrastructure; D007633:Keratins / Q000276:immunology; D007668:Kidney / Q000276:immunology / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000276:immunology; D051379:Mice; D018396:Mucin-1; D014021:Tissue Preservation / Q000379:methods*; D014404:Tubulin / Q000276:immunology; D014407:Tumor Cells, Cultured
D005404:Fixatives; D008562:Membrane Glycoproteins; D018396:Mucin-1; D014404:Tubulin; D007633:Keratins; D003094:Collagen