Abstract
Immunogold labeling of freeze-fracture replicas has recently been used for high-resolution visualization of protein localization in electron microscopy. This method has higher labeling efficiency than conventional immunogold methods for membrane molecules allowing precise quantitative measurements. However, one of the limitations of freeze-fracture replica immunolabeling is difficulty in keeping structural orientation and identifying labeled profiles in complex tissues like brain. The difficulty is partly due to fragmentation of freeze-fracture replica preparations during labeling procedures and limited morphological clues on the replica surface. To overcome these issues, we introduce here a grid-glued replica method combined with SEM observation. This method allows histological staining before dissolving the tissue and easy handling of replicas during immunogold labeling, and keeps the whole replica surface intact without fragmentation. The procedure described here is also useful for matched double-replica analysis allowing further identification of labeled profiles in corresponding P-face and E-face.
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Acknowledgments
We thank Prof. Elek Molnár for providing us a pan-AMPAR antibody used in Fig. 2 and Dr. Ludek Lovicar for technical assistance in scanning electron microscope imaging. This work was supported by the European Union (HBP—Project Ref. 604102).
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Harada, H., Shigemoto, R. (2016). Immunogold Protein Localization on Grid-Glued Freeze-Fracture Replicas. In: Schwartzbach, S., Skalli, O., Schikorski, T. (eds) High-Resolution Imaging of Cellular Proteins. Methods in Molecular Biology, vol 1474. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6352-2_12
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DOI: https://doi.org/10.1007/978-1-4939-6352-2_12
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