The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method
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: vimentinpost-fixationparaformaldehydealcohol/acetic acidmethanol
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