Improved method to retain cytosolic reporter protein fluorescence while staining for nuclear proteins

Cytometry A. 2014 Jul;85(7):621-7. doi: 10.1002/cyto.a.22451. Epub 2014 Feb 19.

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.

Keywords: EGFP; EYFP; FoxP3; RFP; RORγt; T-bet; fate mapping; fixation; flow cytometry; formaldehyde; mouse T cells.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cytokines / analysis*
  • Cytoplasm / metabolism
  • Fixatives
  • Flow Cytometry / methods*
  • Fluorescent Dyes
  • Forkhead Transcription Factors
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Mycobacterium tuberculosis / immunology
  • Nuclear Proteins / analysis*
  • Nuclear Receptor Subfamily 1, Group F, Member 3
  • Staining and Labeling
  • T-Box Domain Proteins
  • T-Lymphocytes / cytology
  • Tissue Fixation / methods
  • Transcription Factors / analysis*

Substances

  • Cytokines
  • Fixatives
  • Fluorescent Dyes
  • Forkhead Transcription Factors
  • Foxp3 protein, mouse
  • Nuclear Proteins
  • Nuclear Receptor Subfamily 1, Group F, Member 3
  • T-Box Domain Proteins
  • T-box transcription factor TBX21
  • Transcription Factors