Correlative immunofluorescence and electron microscopy on the same section of epon-embedded material

Area of Science:

• Cell Biology
• Microscopy
• Virology

Background:

• Correlative microscopy combines different imaging techniques to provide comprehensive cellular information.
• High-resolution ultrastructural localization of viral proteins is crucial for understanding infection mechanisms.

Purpose of the Study:

• To develop and validate a rapid, same-section correlative immunofluorescence-electron microscopy (IF-EM) method.
• To demonstrate the ultrastructural association of wound tumor virus (WTV) core protein with cellular inclusions.

Main Methods:

• Semithick (0.25-0.50 micron) cell sections were stained with fluorescein isothiocyanate (FITC)-conjugated antibodies.
• Sections were embedded in Epon and viewed using high-resolution immunofluorescence microscopy.
• The same sections were subsequently stained with uranyl and lead for electron microscopy (EM).
• Method specificity was confirmed by colocalizing tubulin and microtubules.

Main Results:

• The technique provided high-resolution immunofluorescence patterns against a non-autofluorescent background.
• Specificity was validated by correlating tubulin immunofluorescence with microtubule distribution observed via EM.
• The 57 kD core protein of wound tumor virus was ultrastructurally localized to two distinct cellular inclusions in infected AC-20 cells.
• The correlative approach demonstrated high fidelity between antigen distribution and ultrastructure.

Conclusions:

• This same-section IF-EM technique offers a rapid and simplified approach for ultrastructural antigen localization.
• It eliminates the need for more complex labeling procedures in many cases.
• The method facilitates the study of viral protein-host cell interactions at the ultrastructural level.