Comparison of fixation protocols for adherent cultured cells applied to a GFP fusion protein of the epidermal growth factor receptor

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Abstract
Background: The analysis of the subcellular distribution of proteins is essential for the understanding of processes such as signal transduction. In most cases, the parallel analysis of multiple components requires fixation and immunofluorescence labeling. Therefore, one has to ascertain that the fixation procedure preserves the in vivo protein distribution. Fusion proteins with the green fluorescent protein (GFP) are ideal tools for this purpose. However, one must consider specific aspects of the fluorophore formation or degradation, i.e. reactions that may interfere with the detection of GFP fusion proteins. Methods: Fusion proteins of the epidermal growth factor receptor (EGFR) with GFP as well as free, soluble GFP stably or transiently expressed in adherent cultured cells served as test cases for comparing the distribution in vivo with that after fixation by conventional epifluorescence and laser scanning microscopy. Indirect immunofluorescence was employed to compare the distributions of the GFP signal and of the GFP polypeptide in the fusion protein. Results: Paraformaldehyde (PFA) fixation with subsequent mounting in the antifading agent Mowiol, but not in Tris‐ or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinuclear region. The redistribution was confirmed with the GFP and EGFR immunofluorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, which also retained the fluorescence of soluble GFP. The anti‐GFP antiserum was negative for the N‐terminal fusion protein. Conclusions: The combined PFA/methanol protocol is universally applicable for the fixation of transmembrane and soluble cytoplasmic proteins and preserves the fluorescence of GFP. Cytometry 35:353–362, 1999.