title: Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins.
abstract: Staining of transcription factors (TFs) together with retention of fluorescent reporter proteins is hindered by loss of fluorescence using current available methods. In this study, it is shown that current TF staining protocols do not destroy fluorescent proteins (FPs) but rather that fixation is not sufficient to retain FPs in the cytosol of the permeabilized cells. In this article, a simple and reliable protocol is elaborated, which allows efficient TF and cytokine staining while retaining FPs inside fixed cells.
authors.affiliation: Institute for Molecular Medicine, University Medical Center of the Johannes Gutenberg, University of Mainz, 55131, Mainz, Germany.
authors.forename: André P
authors.initials: AP
authors.lastname: Heinen
authors.forename: Florian
authors.initials: F
authors.lastname: Wanke
authors.forename: Sonja
authors.initials: S
authors.lastname: Moos
authors.forename: Sebastian
authors.initials: S
authors.lastname: Attig
authors.forename: Hervé
authors.initials: H
authors.lastname: Luche
authors.forename: Prajna Paramita
authors.initials: PP
authors.lastname: Pal
authors.forename: Nediljko
authors.initials: N
authors.lastname: Budisa
authors.forename: Hans Jörg
authors.initials: HJ
authors.lastname: Fehling
authors.forename: Ari
authors.initials: A
authors.lastname: Waisman
authors.forename: Florian C
authors.initials: FC
authors.lastname: Kurschus
mesh_terms: D000818:Animals; D016207:Cytokines / Q000032:analysis*; D003593:Cytoplasm / Q000378:metabolism; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D051858:Forkhead Transcription Factors; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009169:Mycobacterium tuberculosis / Q000276:immunology; D009687:Nuclear Proteins / Q000032:analysis*; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3; D013194:Staining and Labeling; D020825:T-Box Domain Proteins; D013601:T-Lymphocytes / Q000166:cytology; D016707:Tissue Fixation / Q000379:methods; D014157:Transcription Factors / Q000032:analysis*; D000098815:T-bet Transcription Factor
chemical_list: D016207:Cytokines; D005404:Fixatives; D005456:Fluorescent Dyes; D051858:Forkhead Transcription Factors; C498833:Foxp3 protein, mouse; D009687:Nuclear Proteins; D057132:Nuclear Receptor Subfamily 1, Group F, Member 3; D020825:T-Box Domain Proteins; D000098815:T-bet Transcription Factor; D014157:Transcription Factors
keywords: EGFP; EYFP; FoxP3; RFP; RORγt; T-bet; fate mapping; fixation; flow cytometry; formaldehyde; mouse T cells