Immunocytochemistry of lipids: chemical fixatives have dramatic effects on the preservation of tissue lipids.
We report here the effects of chemical fixatives on lipids studied under conditions simulating the immunogold labelling of phosphatidylserine. Using anti-phosphatidylserine antibodies, it is shown that the labelling intensity of a phosphatidylserine/phosphatidylcholine coating depends largely on the conditions of fixation. In fact, the usual aldehydic fixatives washed out most of the phostphatidylserine, thus preventing the binding of anti-phosphatidylserine antibodies. This was confirmed on biological samples such as rat liver and brain by measuring the loss of radiolabelled lipids during the fixation procedure. Furthermore, the complete procedure of tissue preparation for electron microscopical observation was investigated. The loss of (radiolabelled) lipids was studied in tissue samples during fixation and resin embedding. The results showed that the classical procedure (glutaraldehyde fixation followed by epoxy resin embedding) results in the loss of 73-91% of the tissue lipids whereas in unfixed, freeze-substituted samples, more than 76% of the tissue lipids are preserved.
Maneta-Peyret
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Laboratoire de Biogenèse Membranaire-Université Victor Segalen Bordeaux 2, CNRS-UMR 5544, France.
Compère
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Moreau
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Goffinet
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Cassagne
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C
D000818:Animals; D000941:Antigens / Q000378:metabolism; D001921:Brain / Q000378:metabolism; D005404:Fixatives / Q000009:adverse effects*; D017110:Freeze Substitution; D007150:Immunohistochemistry / Q000379:methods*; D050356:Lipid Metabolism; D008055:Lipids / Q000737:chemistry*; D008099:Liver / Q000378:metabolism; D008854:Microscopy, Electron; D010718:Phosphatidylserines / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D016622:Silver Staining; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation / Q000379:methods*; D014316:Tritium
D000941:Antigens; D005404:Fixatives; D008055:Lipids; D010718:Phosphatidylserines; D014316:Tritium