Cite as: Cold Spring Harb. Protoc.; 2008; doi:10.1101/pdb.prot5018

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Immunogold Staining of Ultrathin Thawed Cryosections for Transmission Electron Microscopy (TEM)

Jeremy N. Skepper and Janet M. Powell

This protocol was adapted from "Ultrastructural Immunochemistry," Chapter 7, in Immunohistochemistry: Methods Express (ed. Renshaw), from the Methods Express series. Scion Publishing Ltd., Oxfordshire, UK, 2006.


INTRODUCTION

A pre-embedding method of immunochemical staining is used if antigens are damaged by resin embedding, or if the best preservation of membranes is required. Applying immunogold reagents to sections of lightly fixed tissue, free of embedding medium, can be a very sensitive method of immunochemical staining. Cells or tissues are fixed as strongly as possible and then treated with a cryoprotectant, which is usually a mixture of sucrose and polyvinylpyrrolidone (PVP). They are frozen onto pins in liquid nitrogen and sectioned at approximately -100°C. The frozen sections are thaw-mounted on to Formvar/nickel film grids and the cryoprotectant is removed by floating the grids on drops of phosphate-buffered saline (PBS). The immunogold staining is performed on the unembedded sections, which are subsequently contrast counterstained and infiltrated with a mixture of methylcellulose and uranyl acetate. In this protocol, samples are sectioned at low temperature, thaw-mounted onto film grids, immunochemically stained, contrast counterstained, and embedded/encapsulated in situ on the grid before viewing by transmission electron microscopy (TEM).


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