An innovative fixative for cytoskeletal components allows high resolution in colocalization studies using immunofluorescence techniques

Abstract

Using a new fixation solution, CytoSkelFix, it is now possible to obtain superior fixation and thus resolution of cytoskeletal components using immunofluorescence and fluorescence microscopy. This fixative combines rapid cell penetration and cellular crosslinking of proteins such that both preservation and resolution of cellular proteins can be detected. The cytoskeleton has proven very difficult to preserve, partly because of the lability of one of the filament systems (microtubules), and one single fixative is incapable of properly preserving microtubules, microfilaments, and intermediate filaments for localization in the same cell. Further, the motor proteins associated with the cytoskeletal elements are even more difficult to preserve, particularly simultaneously with the fiber system with which they associate. We present evidence that CytoSkelFix is a superior preservative and would be useful in fixation for all types of immunofluorescence colocalization studies where superior preservation is required.

Migration of bone marrow stromal cells in 3D: 4 Color methodology reveals spatially and temporally coordinated events

“…In addition, each network is in the presence of its monomers in the cytoplasm and is partly masked by a variety of associated soluble and insoluble proteins which can complicate immunocytochemistry procedures [Arcangeletti et al, 1997]. Limiting factors for 3D resolution of cytoskeletal elements in immunofluorescence lies in the concurrent achievement of good structural preservation and detailed labeling [Robinson and Snyder, 2004]. Most cytological processing methods are not optimized for 3D cell preservation, and therefore do not harness the full potential of confocal microscopy to assess cell structures representative of the physiological steady state [Arcangeletti et al, 1997].…”

Glyoxal Does Not Preserve Cellular Proteins as Accurately as PFA: A Microscopical Survey of Epitopes

“…First introduced in 1893, formaldehyde (FA), as a member of aldehyde fixatives, in the form of a water-based, diluted solution of 1:10 has been the most commonly-used chemical fixative, and it is found in many fixative cocktails (Marcon, Bressenot et al, 2009). Paraformaldehyde (PFA), the polymerized form of FA, is generally favoured over FA because PFA crosslinks amino groups without altering the tertiary structure of proteins, thereby cellular epitopes remain relatively well-preserved in a successful labelling protocol with specific antibodies (Fujiwara, 1980, Leyton-Puig, Kedziora et al, 2016, Robinson & Snyder, 2004. PFA fixation has been widely adopted to preserve cell morphology for immunolabeling where the final concentration of PFA in the fixative solution is around 3-4% (Kim, Kim et al, 2017).…”

Recent progress in histochemistry and cell biology: the state of the art 2005

“…Their protocol utilized formaldehyde fixation followed by permeabilization with digitonin or saponin for the detection of immunoreactivity, suggesting that lipid droplet proteins are likely solubilized by strong detergents. Robinson and Snyder (2004) tested successfully a new commercially available fixative for immunohistochemical detection of various cytoskeletal proteins and associated motor proteins and demonstrated its superiority compared with existing fixatives. In particular, improvements for double immunofluorescence labeling were reported.…”