Cytoplasmic microtubular images in glutaraldehyde-fixed tissue culture cells by electron microscopy and by immunofluorescence microscopy.
Electron microscopy and indirect immunofluorescence microscopy using monospecific tubulin antibodies were performed in parallel on glutaraldehyde-fixed tissue culture cells without osmium fixation. In order to reduce the excess aldehyde groups of the strongly crosslinked cellular matrix, which normally interfere with subsequent immunofluorescence microscopy, a mild NaBH(4) treatment was introduced during or after the dehydration steps. Cells processed through the NaBH(4) step show, in transmission electron microscopy, normal cytoplasmic microtubules approximately 250 A in diameter. When such cells are subjected to indirect immunofluorescence microscopy using monospecific tubulin antibody they reveal a complex system of unbroken, fine, fluorescent fibers traversing the cytoplasm between the perinuclear space and the plasma membrane. Thin sections of cells processed through the indirect immunofluorescence procedure show antibody-decorated microtubules with a diameter of approximately 600 A. This decoration is not obtained when non-immune IgGs are used instead of monospecific antitubulin IgGs. Thus, a direct comparison of cytoplasmic microtubules in glutaraldehyde-fixed cells by both electron microscopy and immunofluorescence microscopy can be obtained.
D002478:Cells, Cultured; D003593:Cytoplasm / Q000648:ultrastructure; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005976:Glutaral; D008854:Microscopy, Electron / Q000379:methods; D008870:Microtubules / Q000648:ultrastructure*; D014404:Tubulin / Q000276:immunology
D005404:Fixatives; D014404:Tubulin; D005976:Glutaral