Microscopy and Microanalysis



Immunolabeling for Correlative Light and Electron Microscopy on Ultrathin Cryosections


Irawati K.  Kandela  a1 , Reiner  Bleher  a2 and Ralph M.  Albrecht  a1 a2 a3 c1
a1 Department of Pharmaceutical Sciences, University of Wisconsin, Madison, WI 53705, USA
a2 Department of Animal Sciences, University of Wisconsin, Madison, WI 53706, USA
a3 Department of Pediatrics, University of Wisconsin, Madison, WI 53706, USA

Article author query
kandela ik   [PubMed][Google Scholar] 
bleher r   [PubMed][Google Scholar] 
albrecht rm   [PubMed][Google Scholar] 

Abstract

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.

(Received August 27 2007)
(Accepted September 11 2007)


Key Words: correlative labeling; immunofluorescence; immuno-EM; colloidal gold particles; fluorophores; cryosections; nanoparticles.

Correspondence:
c1 Corresponding author. E-mail: rmalbrec@facstaff.wisc.edu