Perfusion fixation preserves enhanced yellow fluorescent protein and other cellular markers in lymphoid tissues

• Authors: Joseph G. Dauner, Emory University; Craig P. Chappell, University of Washington; Ifor Williams, Emory University; Joshy Jacob, Emory University
• Journal Title: Journal of Immunological Methods
• Volume: Volume 340, Number 2
• Publisher: Elsevier | 2009-01-30, Pages 116-122
• Type of Work: Article | Post-print: After Peer Review
• Publisher DOI: http://doi.org/10.1016/j.jim.2008.10.010
• Subjects: Health Sciences, Immunology; Health Sciences, Pathology
• Keywords: Science & Technology; Life Sciences & Biomedicine; Biochemical Research Methods; Immunology; Biochemistry & Molecular Biology; EYFP; Immunofluorescence microscopy; Lymphoid organs; Tissue sections; IN-VIVO; TRANSGENIC MICE; MOUSE MODEL; T-CELLS; EXPRESSION; MEMORY; LOCALIZATION; MICROSCOPY; AEQUOREA; PROMOTER

Abstract

Fluorescent proteins are increasingly being used to analyze cellular gene expression and to facilitate tracking of cell lineages in vivo. One of these, enhanced yellow fluorescent protein (EYFP) has several properties such as intense fluorescence and little to no toxicity in cells, which makes it an excellent molecule to label proteins and cells of interest. In live cells, visualization of EYFP has been highly successful; however, detection of EYFP in lymphoid tissue sections, particularly in combination with other markers of interest has been difficult. This is because of the enhanced solubility of EYFP in the absence of fixation. When extended fixation protocols are employed, EYFP is preserved but detection of other cellular antigens becomes problematic due to over fixation. Here we demonstrate that EYFP-expressing T and B cells can be efficiently visualized in lymphoid tissue sections without compromising the ability to detect other cellular markers.