Preservation and immunogold localization of lipids by freeze-substitution and low temperature embedding.
The success of post-embedding immunocytochemistry depends largely on the preparation methods. The requirements for structural preservation and immunocytochemistry are in some cases contradictory. This is especially the case in the study of lipid-rich structures and the localization of lipid components. Earlier work on freeze-substitution has shown that this method is very promising for the preservation of lipids and the immunocytochemical localization of lipids at the electron microscopical level. In this study we show that freeze-substitution in combination with low temperature embedding in Lowicryl HM20 has fulfilled this promise. Lamellar bodies in alveolar type II cells contain about 90% lipids and are very difficult to preserve in ultrathin cryosections. Lowicryl sections of freeze-substituted lung tissue shows excellent preservation of lamellar bodies in combination with immunogold localization of a hydrophobic surfactant protein. With an antibody against the Forssman glycolipid we demonstrate a highly reproducible intracellular localization of this glycolipid with high specificity and resolution. This method results in the retention of lipids and glycolipids and allows postembedding immunogold labeling.
Voorhout
W
W
Department of Cell Biology, Medical School, University of Utrecht, The Netherlands.
van Genderen
I
I
van Meer
G
G
Geuze
H
H
D000180:Acrylic Resins; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D015925:Cryopreservation; D004285:Dogs; D005577:Forssman Antigen / Q000032:analysis; D005915:Globosides / Q000032:analysis*; D006801:Humans; D007150:Immunohistochemistry; D007668:Kidney / Q000648:ultrastructure*; D008168:Lung / Q000648:ultrastructure*; D016610:Tissue Embedding; D014021:Tissue Preservation
D000180:Acrylic Resins; D005915:Globosides; C010163:Forssman glycolipid; C070811:Lowicryl HM-20; D005577:Forssman Antigen