GFP immunogold staining, from light to electron microscopy, in mammalian cells

GFP has emerged as an important reporter for monitoring gene expression, protein localization, cell transformation and cell lineage. The development of GFP as a marker in many different biological systems has emphasized the need to image GFP at high resolution. GFP immunogold labeling with colloidal gold particles becomes essential for electron microscopy (EM) ultrastructural detection. Because of the small size, colloidal gold particles require silver enhancement, a procedure to increase the size of the particle as well as gold toning to stabilize the silver layer. GFP preembedding immunogold staining enables high quality cellular-ultrastructural EM analysis mainly for two reasons, on one hand it allows adequate fixation for EM analysis maintaining GFP antigenicity, on the other hand it also enables the epoxy resins inclusion after immunogold staining. Both of them help to preserve better the ultrastructure. However GFP immunogold staining presents some drawbacks, such as the progressive decrease in immunogold labeling with tissue depth. Special attention must be taken when using GFP-tagged protein, since the fusion could interfere with their localization and function. In this review we provide a detailed protocol of the GFP immunogold staining, their main applications for EM and possible troubles.

Graphical abstractFigure optionsDownload full-size imageDownload high-quality image (64 K)Download as PowerPoint slideHighlights► We review the use of GFP immunogold staining for electron microscopy detection. ► Reporter gene, fusion tag and cell marker are the main applications of GFP immunogold-staining. ► A detailed protocol for GFP immunogold staining is provided. ► Pre-embedding GFP immunogold staining preserves better the ultrastructure. ► The low immunogold penetration and the interference with the localization of GFP-tagged proteins are some disadvantages of the GFP immunogold staining.