Biochemical, electron microscopic and immunohistological observations of cationic detergent-extracted cells: detection and improved preservation of microextensions and ultramicroextensions.
BACKGROUND
Filopodia, retraction fibers and microvilli, are fragile microextensions of the plasma membrane that are easily damaged by mechanical force during specimen preparation for microscopy. To preserve these structures for electron microscopy glutaraldehyde is generally used, but it often causes antigen masking. By contrast, formaldehyde is generally used for immunofluorescence light microscopy, but few studies have been concerned with the loss of microextensions.


RESULTS
We demonstrate in biochemical experiments that cultured cells needed to be kept in 4% formaldehyde for at least 60 min at room temperature or for 20 min at 37 degrees C to irreversibly crosslink most of the polypeptides. Also, fragmentation of fragile microextensions was observed after Triton X-100 extraction depending on concentration and extent of crosslinking. We also report on a novel fixation procedure that includes the cationic detergent dodecyltrimethylammonium chloride (DOTMAC). Treatment of NIH3T3 cells with DOTMAC resulted in complete removal of membrane lipids and in good preservation of the cytoskeleton in microextensions as well as preservation of ultramicroextensions of <0.05 microm in diameter that have not been observed previously unless glutaraldehyde was used. Stress fibers and microextensions of DOTMAC-extracted cells were readily stained with anti-beta-actin antibodies, and antibodies to vinculin and moesin stained focal contacts and microextensions, respectively.


CONCLUSIONS
Some microextensions were fragmented by the standard Triton X-100 permeabilization method. By contrast, DOTMAC completely extracted membrane lipids while maintaining the cytoskeleton of microextensions. Thus, DOTMAC treatment may provide a valuable new tool for the reliable visualization of previously undetectable or poorly detectable antigens while preserving the actin cytoskeleton of microextensions.
Nakamura
F
F
Laboratory of Environmental Biochemistry, Department of Environmental Biology, Graduate School of Agricultural Science, Tohoku University, Sendai 981-8555, Japan. fnakamura@rics.bwh.havard.edu
D016475:3T3 Cells; D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000818:Animals; D002412:Cations; D002457:Cell Extracts / Q000032:analysis; D022081:Cell Surface Extensions / Q000648:ultrastructure*; D003598:Cytoskeletal Proteins / Q000032:analysis / Q000276:immunology; D003599:Cytoskeleton / Q000648:ultrastructure; D003902:Detergents / Q000737:chemistry*; D005404:Fixatives / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D007150:Immunohistochemistry; D051379:Mice; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008870:Microtubules / Q000648:ultrastructure; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification; D011108:Polymers / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry*; D016707:Tissue Fixation / Q000379:methods; D014021:Tissue Preservation / Q000379:methods*
D002412:Cations; D002457:Cell Extracts; D003598:Cytoskeletal Proteins; D003902:Detergents; D005404:Fixatives; D010455:Peptides; D011108:Polymers; D000644:Quaternary Ammonium Compounds; D005557:Formaldehyde; C022806:dodecyltrimethylammonium; C003043:paraform