TY - JOUR
T1 - Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles
AU - Kandela, Irawati K.
AU - Bleher, Reiner
AU - Albrecht, Ralph M.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2007/10
Y1 - 2007/10
N2 - Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscoopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and α-actinin bands of skeletal muscle tissue and also actin and α-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.
AB - Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscoopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric miscroscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and α-actinin bands of skeletal muscle tissue and also actin and α-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM.
KW - Colloidal gold nanoparticles
KW - Colloidal metal nanoparticles
KW - Colloidal palladium nanoparticles
KW - Correlative immunolabeling
KW - Fluorophores
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U2 - 10.1369/jhc.6A7124.2007
DO - 10.1369/jhc.6A7124.2007
M3 - Article
C2 - 17652267
AN - SCOPUS:34548714245
SN - 0022-1554
VL - 55
SP - 983
EP - 990
JO - Journal of Histochemistry and Cytochemistry
JF - Journal of Histochemistry and Cytochemistry
IS - 10
ER -