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The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method
Affiliations- University of Health Sciences, Ankara, Türkiye
- Inonu University
open access
Abstract
Background: Post-fixation of sections is especially required for cryostat sections of fresh frozen tissues. Vimentin is an intermediate filament in both fibrillary and non-fibrillary form, expressed in Müller’s cells and astrocytes of the retina. Our aim was to determine the best post-fixation method for visualising vimentin in archival mouse eyes.
Materials and methods: We used an archival mouse eye, slightly pre-fixed with paraformaldehyde and stored at –80°C for 4 years. We tried three fixatives (paraformaldehyde [PFA], alcohol/acetic acid [AAA] and methanol) for post-fixation of eye sections.
Results: We showed that post-fixation alters the labelling properties of vimentin expressed in the retina. In the sections with no post-fixation, vimentin positivity was observed in and around the nuclei in non-fibrillary form. In PFA post-fixed sections, the vimentin in the retina was not observed as fibrils. Positivity was observed in the nuclei and in perinuclear regions of the cells. In AAA post-fixed sections, positive labelling was observed around the nuclei as fibrils. In methanol post-fixed sections, labelling was observed around the nuclei as fibrils.
Conclusions: We conclude that post-fixation with AAA is more convenient for immunofluorescent labelling of vimentin in the retina for slightly PFA pre-fixed and long-term stored retina. (Folia Morphol 2018; 77, 2: 246–252)
Abstract
Background: Post-fixation of sections is especially required for cryostat sections of fresh frozen tissues. Vimentin is an intermediate filament in both fibrillary and non-fibrillary form, expressed in Müller’s cells and astrocytes of the retina. Our aim was to determine the best post-fixation method for visualising vimentin in archival mouse eyes.
Materials and methods: We used an archival mouse eye, slightly pre-fixed with paraformaldehyde and stored at –80°C for 4 years. We tried three fixatives (paraformaldehyde [PFA], alcohol/acetic acid [AAA] and methanol) for post-fixation of eye sections.
Results: We showed that post-fixation alters the labelling properties of vimentin expressed in the retina. In the sections with no post-fixation, vimentin positivity was observed in and around the nuclei in non-fibrillary form. In PFA post-fixed sections, the vimentin in the retina was not observed as fibrils. Positivity was observed in the nuclei and in perinuclear regions of the cells. In AAA post-fixed sections, positive labelling was observed around the nuclei as fibrils. In methanol post-fixed sections, labelling was observed around the nuclei as fibrils.
Conclusions: We conclude that post-fixation with AAA is more convenient for immunofluorescent labelling of vimentin in the retina for slightly PFA pre-fixed and long-term stored retina. (Folia Morphol 2018; 77, 2: 246–252)
Keywords
vimentin, post-fixation, paraformaldehyde, alcohol/acetic acid, methanol
About this articleTitle
The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method
Journal
Issue
Article type
Original article
Pages
246-252
Published online
2017-08-31
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2699
Article views/downloads
1628
DOI
Pubmed
Bibliographic record
Folia Morphol 2018;77(2):246-252.
Keywords
vimentin
post-fixation
paraformaldehyde
alcohol/acetic acid
methanol
Authors
B. Baykal
C. Korkmaz
N. Kocabiyik
O.M. Ceylan
References (21)- Alves VA, Wakamatsu A, Kanamura CT, et al. [The importance of fixation in immunohistochemistry: distribution of vimentin and cytokeratins in samples fixed in alcohol and formol]. Rev Hosp Clin Fac Med Sao Paulo. 1992; 47(1): 19–24.
- Boon ME, Kok LP. Formalin is deleterious to cytoskeleton proteins: do we need to replace it by formalin-free Kryofix? Eur J Morphol. 1991; 29(3): 173–180.
- Dahl D, Bignami A, Weber K, et al. Filament proteins in rat optic nerves undergoing Wallerian degeneration: localization of vimentin, the fibroblastic 100-A filament protein, in normal and reactive astrocytes. Exp Neurol. 1981; 73(2): 496–506.
- Dahl D, Rueger DC, Bignami A, et al. Vimentin, the 57 000 molecular weight protein of fibroblast filaments, is the major cytoskeletal component in immature glia. Eur J Cell Biol. 1981; 24(2): 191–196.
- Franke WW, Schmid E, Osborn M, et al. Different intermediate-sized filaments distinguished by immunofluorescence microscopy. Proc Natl Acad Sci U S A. 1978; 75(10): 5034–5038.
- Fritschy JM. Is my antibody-staining specific? How to deal with pitfalls of immunohistochemistry. Eur J Neurosci. 2008; 28(12): 2365–2370.
- Gilmer B, Kilkenny J, Tomaszewski C, et al. Hyperbaric oxygen does not prevent neurologic sequelae after carbon monoxide poisoning. Acad Emerg Med. 2002; 9(1): 1–8.
- Granger BL, Lazarides E. Desmin and vimentin coexist at the periphery of the myofibril Z disc. Cell. 1979; 18(4): 1053–1063.
- Guidry C, Medeiros NE, Curcio CA. Phenotypic variation of retinal pigment epithelium in age-related macular degeneration. Invest Ophthalmol Vis Sci. 2002; 43(1): 267–273.
- Hartig R, Shoeman RL, Janetzko A, et al. DNA-mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells. J Cell Sci. 1998; 111 (Pt 24): 3573–3584.
- Helfand BT, Mendez MG, Murthy SN, et al. Vimentin organization modulates the formation of lamellipodia. Mol Biol Cell. 2011; 22(8): 1274–1289.
- Hoetelmans RW, Prins FA, Cornelese-ten Velde I, et al. Effects of acetone, methanol, or paraformaldehyde on cellular structure, visualized by reflection contrast microscopy and transmission and scanning electron microscopy. Appl Immunohistochem Mol Morphol. 2001; 9(4): 346–351.
- Johansson UE, Eftekhari S, Warfvinge K. A battery of cell- and structure-specific markers for the adult porcine retina. J Histochem Cytochem. 2010; 58(4): 377–389.
- Lewis GP, Fisher SK. Müller cell outgrowth after retinal detachment: association with cone photoreceptors. Invest Ophthalmol Vis Sci. 2000; 41(6): 1542–1545.
- Nakazawa T, Takeda M, Lewis GP, et al. Attenuated glial reactions and photoreceptor degeneration after retinal detachment in mice deficient in glial fibrillary acidic protein and vimentin. Invest Ophthalmol Vis Sci. 2007; 48(6): 2760–2768.
- Rehak M, Hollborn M, Iandiev I, et al. Retinal gene expression and Müller cell responses after branch retinal vein occlusion in the rat. Invest Ophthalmol Vis Sci. 2009; 50(5): 2359–2367.
- Ross M, Pawlina W. Eye. In: Histology: A Text and Atlas with Correlated Cell and Molecular Biology. Lippincott Williams & Wilkins, China. 2011: 896–908.
- Rustad OJ, Kaye V, Cerio R, et al. Postfixation of cryostat sections improves tumor definition in Mohs surgery. J Dermatol Surg Oncol. 1989; 15(12): 1262–1267.
- Schnitzer J. Chapter 7 Astrocytes in mammalian retina. Progress in Retinal Research. 1988; 7: 209–231.
- Sethi CS, Lewis GP, Fisher SK, et al. Glial remodeling and neural plasticity in human retinal detachment with proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci. 2005; 46(1): 329–342.
- van Harreveld A, Khattab FI. Perfusion fixation with glutaraldehyde and post-fixation with osmium tetroxide for electron microscopy. J Cell Sci. 1968; 3(4): 579–594.
