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

Background: The analysis of the subcellular distribution of proteins is ess ential for the understanding of processes such as signal transduction, In m ost cases, the parallel analysis of multiple components requires fixation a nd immunofluorescence labeling. Therefore, one has to ascertain that the fi xation procedure preserves the in vivo protein distribution. Fusion protein s 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) wit h GFP as well as free, soluble GFP stably or transiently expressed in adher ent cultured cells served as test cases for comparing the distribution in v ivo with that after fixation by conventional epifluorescence and laser scan ning microscopy. Indirect immunofluorescence was employed to compare the di stributions of the GFP signal and of the GFP polypeptide in the fusion prot ein. Results: Paraformaldehyde (PFA) fixation with subsequent mounting in the an tifading agent Mowiol, but not in Tris- or HEPES buffered saline, led to a partial redistribution of the EGFR from the plasma membrane to the perinucl ear region. The redistribution was confirmed with the GFP and EGFR immunofl uorescence. The in vivo distribution in Mowiol mounted cells was preserved if cells were treated with a combined PFA/methanol fixation procedure, whic h 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 f or the fixation of transmembrane and soluble cytoplasmic proteins and prese rves the fluorescence of GFP. (C) 1999 Wiley-Liss, Inc.