Immunolabeling artifacts and the need for live-cell imaging

Abstract

Fluorescent fusion proteins have revolutionized examination of proteins in living cells. Still, studies using these proteins are met with criticism because proteins are modified and ectopically expressed, in contrast to immunofluorescence studies. However, introducing immunoreagents inside cells can cause protein extraction or relocalization, not reflecting the in vivo situation. Here we discuss pitfalls of immunofluorescence labeling that often receive little attention and argue that immunostaining experiments in dead, permeabilized cells should be complemented with live-cell imaging when scrutinizing protein localization.

A near-infrared fluorophore for live-cell super-resolution microscopy of cellular proteins

“…All nanoscopy approaches rely on switching between the fluorescent and dark states of dyes 17 ; thus, in such experiments, the photophysical properties of the fluorophore critically affect the attainable resolution 18 . Furthermore, ideally nanoscopy of biological structures is performed on living cells, as this avoids the introduction of structural artefacts during fixation of the cells and permits characterization of the dynamic processes 19 .…”

Right on Q: Genetics begin to Unravel Coxiella Burnetii host cell Interactions

“…Effectors target host systems with defined roles in Coxiella cellular parasitism, and identifying their subcellular destinations can help design laboratory inquiries into function. However, detection of native effectors is exceedingly difficult; thus, ectopic expression of effector-fluorescent protein chimeras has been extensively used for trafficking studies, despite known artifacts of this procedure [120]. Effectors traffic to mitochondria [87,92,94,121], endoplasmic reticulum [91,94,96,122], Golgi apparatus [87], lysosomes [87,92,107,122], autophagosomes [91,93], endocytic vesicles [73,107], nucleus [87,94,[121][122][123], microtubules [92,122] and ubiquitinated proteins [91,93].…”

Cytokinetic Abscission: Phosphoinositides and ESCRTs Direct the Final Cut

“…However fixation processes and cell permeabilization can partially alter PtdIns accessibility and localization, sometimes resulting in mislabelling and unspecific staining. More importantly, this technique precludes the analysis of the dynamic distribution of the lipids in living cells (Schnell et al, 2012). To avoid these problems, alternative pioneering approaches firstly proposed to use PtsIns binding domain as biosensors by their fusion to fluorescent proteins (Stauffer et al, 1998;Varnai and Balla, 1998).…”