Immunogold probes for electron microscopy: evaluation of staining by fluorescence microscopy

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Immunogold probes are crucial for ultrastructural localization in electron microscopy.
• Evaluating immunogold probe staining requires time-consuming electron microscopy processing.
• Optimizing staining parameters for immunogold techniques is essential for accurate results.

Purpose of the Study:

• To develop a method for evaluating immunogold probe staining at the light microscopic level.
• To correlate light microscopic observations with subsequent electron microscopic findings.
• To streamline the optimization of immunogold labeling protocols.

Main Methods:

• Cultured Dunning R-3327-H rat prostatic adenocarcinoma cells were fixed with methanol.
• A two-step immunogold technique was employed using primary anti-keratin antibody and gold-labeled secondary antibody.
• Visualization of bound immunogold probe was achieved using a fluorescent tertiary anti-immunogold probe antibody.

Main Results:

• Fluorescence microscopy revealed a typical keratin cytoskeleton in whole cell monolayers.
• Subsequent electron microscopy confirmed cytoplasmic intermediate filaments decorated with colloidal gold.
• The additional fluorescent staining step did not impede subsequent electron microscopy processing.

Conclusions:

• The developed method allows for light microscopic evaluation of immunogold probe staining.
• This technique facilitates optimization of staining parameters without repeated electron microscopy.
• It provides a valuable correlation between light and ultrastructural immunogold labeling patterns.