Immunolabeling for correlative light and electron microscopy on ultrathin cryosections.
Correlative labeling permits colocalization of molecular species for observation of the same sample in light (LM) and electron microscopy (EM). Myosin bands in ultrathin cryosections were labeled using both fluorophore conjugated to secondary antibody (IgG) and colloidal gold (cAu) particles conjugated to primary IgG as reporters for LM and transmission electron microscopy (TEM), respectively. This technique allows rapid evaluation of labeling via LM, prior to more time-consuming observations with TEM and also yields two complementary data sets in one labeling procedure. Quenching of the fluorescent signal was inversely related to the distance between fluorophore and cAu particles. The signal from fluorophore conjugated to secondary antibody was inversely proportional to the size of cAu conjugated to primary antibody. Where fluorophore and cAu were bound to the same antibody, the fluorescence signal was nearly completely quenched regardless of fluorophore excitation or emission wavelength and regardless of particle size, 3 nm and larger. Colloidal metal particles conjugated to primary antibody provide high spatial resolution for EM applications. Fluorophore conjugated to secondary antibody provides spatial resolution well within that of conventional fluorescence microscopy. Use of fluorescent secondary antibody moved the fluorophore a sufficient distance from the cAu particles on the primary antibody to limit quenching of fluorescence.
Kandela
Irawati K
IK
Department of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA.
Bleher
Reiner
R
Albrecht
Ralph M
RM
D000818:Animals; D016616:Cryoultramicrotomy; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes / Q000737:chemistry*; D017979:Gold Colloid / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D051379:Mice; D008853:Microscopy / Q000379:methods; D016253:Microscopy, Immunoelectron / Q000379:methods*; D018482:Muscle, Skeletal / Q000737:chemistry / Q000648:ultrastructure; D009218:Myosins / Q000737:chemistry*; D053758:Nanoparticles / Q000737:chemistry; D051381:Rats; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D017979:Gold Colloid; D007074:Immunoglobulin G; D009218:Myosins