Abstract
Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. However, conventional nanogold labelling requires time-consuming nanogold probe preparation and ultrathin sectioning of cell/tissue samples. Here, we introduce an in situ strategy involving nanogold nucleation in immunoenzymatic products on universal paraffin/cryostat sections and provide unique insight into nanogold development under hot-humid air conditions. Nanogold particles were specifically localized on kidney podocytes to target synaptopodin. Transmission electron microscopy revealed secondary growth and self-assembly that could be experimentally controlled by bovine serum albumin stabilization and phosphate-buffered saline acceleration. Valuable retrospective nanogold labelling for gastric H+/K+-ATPase was achieved on vintage immunoenzymatic deposits after a long lapse of 15 years (i.e., 15-year-old deposits). The present in situ nanogold labelling is anticipated to fill the gap between light and electron microscopy to correlate cell/tissue structure and function.
Highlights
Immunocytochemistry visualizes the exact spatial location of target molecules
This study aimed to develop its application in immunocytochemical localization, which remains challenging owing to difficulties in achieving labeling intensity with large particles
We introduce a new strategy involving in situ nanogold nucleation in immunoenzymatic 3,3’-diaminobenzidine tetrahydrochloride (DAB) products on universal paraffin/cryostat sections and provide novel insight into nanogold development in hot-humid air conditions for ultrastructural localization
Summary
Immunocytochemistry visualizes the exact spatial location of target molecules. The most common strategy for ultrastructural immunocytochemistry is the conjugation of nanogold particles to antibodies as probes. Visualization of the exact location of targeting molecules presents unique spatial information that no other biomedical analysis can provide This process is called immunocytochemistry, a combination of immunochemistry and cell morphology, which uses specific antibodies against the target molecules of interest. A variety of nanogold-conjugated antibodies are commercially available to decrease preparation, but ultrathin sectioning of the cell/tissue samples still requires time and skill for post-embedding nanogold probe labeling. There is a trade-off relation between large particles and labeling intensity in post-embedding nanogold probe labeling[14], but large particles (>30 nm) are indispensable for visualization under low-vacuum SEM In this context, we introduce a new strategy involving in situ nanogold nucleation in immunoenzymatic 3,3’-diaminobenzidine tetrahydrochloride (DAB) products on universal paraffin/cryostat sections and provide novel insight into nanogold development in hot-humid air conditions for ultrastructural localization
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