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The influence of post-fixation on visualising vimentin in the retina using immunofluorescence method
B Baykal1, C Korkmaz, N Kocabiyik
1University of Health Sciences, Ankara, Turkey. baris_baykal@yahoo.com.
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 (pa-raformaldehyde [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).
Insights
Post-fixation methods impact vimentin visualization in archival mouse retinas. Alcohol/acetic acid (AAA) fixation is optimal for immunofluorescent labeling of vimentin in stored eye sections.
Area of Science:
- Ophthalmology
- Neuroscience
- Histology
Background:
- Cryostat sections of fresh frozen tissues require post-fixation.
- Vimentin, an intermediate filament, is present in Müller's cells and astrocytes in the retina.
- Assessing vimentin visualization in archival mouse eyes is crucial.
Purpose of the Study:
- To determine the optimal post-fixation method for visualizing vimentin in archival mouse eyes.
- To evaluate the effects of different post-fixation techniques on vimentin's fibrillar and non-fibrillar forms.
Main Methods:
- Archival mouse eyes, pre-fixed with paraformaldehyde and stored for 4 years, were used.
- Sections were post-fixed using paraformaldehyde (PFA), alcohol/acetic acid (AAA), or methanol.
- Immunofluorescent labeling was employed to visualize vimentin.
Main Results:
- Post-fixation significantly altered vimentin's labeling properties in the retina.
- No post-fixation showed non-fibrillar vimentin around nuclei.
- PFA post-fixation resulted in non-fibrillar vimentin in nuclei/perinuclear regions.
- AAA and methanol post-fixation revealed fibrillar vimentin around nuclei.
Conclusions:
- Alcohol/acetic acid (AAA) post-fixation is most suitable for immunofluorescent vimentin labeling in archival, PFA-pre-fixed mouse retinas.
- The choice of post-fixation impacts the observed vimentin structure (fibrillar vs. non-fibrillar).
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