Immunogold protein localization on grid-glued freeze-fracture replicas Book Section

Author(s): Harada, Harumi; Shigemoto, Ryuichi
Article/Chapter Title: Immunogold protein localization on grid-glued freeze-fracture replicas
Title Series: Methods in Molecular Biology
Affiliation IST Austria
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.
Keywords: Immunogold labeling; Freeze-fracture replica; SDS digestion; scanning electron microscopy, high-resolution analyses
Book Title: High-Resolution Imaging of Cellular Proteins
Volume: 1474
ISBN: 978-1-4939-6352-2
Publisher: Springer  
Date Published: 2016-08-12
Start Page: 203
End Page: 216
DOI: 10.1007/978-1-4939-6352-2_12
Notes: We thank Prof. Elek Molnár for providing us a pan-AMPAR anti-body 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).
Open access: no