A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue.

Détails

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Etat: Public
Version: Final published version
Licence: CC BY 4.0
ID Serval
serval:BIB_A997FAE44083
Type
Article: article d'un périodique ou d'un magazine.
Collection
Publications
Institution
Titre
A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue.
Périodique
Scientific Reports
Auteur(s)
Scandella V., Paolicelli R.C., Knobloch M.
ISSN
2045-2322 (Electronic)
ISSN-L
2045-2322
Statut éditorial
Publié
Date de publication
04/09/2020
Peer-reviewed
Oui
Volume
10
Numéro
1
Pages
14642
Langue
anglais
Notes
Publication types: Journal Article
Publication Status: epublish
Résumé
The green fluorescent protein (GFP) is a powerful reporter protein that allows labeling of specific proteins or entire cells. However, as GFP is a small soluble protein, it easily crosses membranes if cell integrity is disrupted, and GFP signal is lost or diffuse if the specimen is not fixed beforehand. While pre-fixation is often feasible for histological analyses, many molecular biology procedures and new imaging techniques, such as imaging mass spectrometry, require unfixed specimens. To be able to use GFP labeling in tissues prepared for such applications, we have tested various protocols to minimize the loss of GFP signal. Here we show that, in cryocut sections of snap-frozen brain tissue from two GFP reporter mouse lines, leaking of the GFP signal is prevented by omitting the commonly performed drying of the cryosections, and by direct post-fixation with 4% paraformaldehyde pre-warmed at 30-37 °C. Although the GFP staining does not reach the same quality as obtained with pre-fixed tissue, GFP localization within the cells that express it is preserved with this method. This protocol can thus be used to identify GFP positive cells on sections originating from unfixed, cryosectioned tissue.
Pubmed
Open Access
Oui
Création de la notice
14/09/2020 9:53
Dernière modification de la notice
30/04/2021 7:13
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