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

Details

Serval ID
serval:BIB_A997FAE44083
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
A novel protocol to detect green fluorescent protein in unfixed, snap-frozen tissue.
Journal
Scientific Reports
Author(s)
Scandella V., Paolicelli R.C., Knobloch M.
ISSN
2045-2322 (Electronic)
ISSN-L
2045-2322
Publication state
Published
Issued date
04/09/2020
Peer-reviewed
Oui
Volume
10
Number
1
Pages
14642
Language
english
Notes
Publication types: Journal Article
Publication Status: epublish
Abstract
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
Yes
Create date
14/09/2020 9:53
Last modification date
18/09/2020 6:26
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