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.
Laboratorio de Morofología Celular, Centro de Investigación Príncipe Felipe, CIBERNED, Valencia, Spain.
M Salomé
MS
Sirerol-Piquer
Arantxa
A
Cebrián-Silla
Clara
C
Alfaro-Cervelló
Ulises
U
Gomez-Pinedo
Mario
M
Soriano-Navarro
José-Manuel García
JM
Verdugo
D000818:Animals; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods*
D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins