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An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

Sun N, Shibata B, Hess JF, FitzGerald PG - Mol. Vis. (2015)

Bottom Line: Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment.The approach shows a notable improvement in preservation of immunoreactivity.On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, CA.

ABSTRACT

Purpose: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.

Methods: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.

Results: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.

Conclusions: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

No MeSH data available.


Related in: MedlinePlus

Retinal labeling with anti-vimentin antibody. All of the freeze substituted samples (A-E) show a higher level of immunoreactivity than the formalin-fixed sample (F). Of all the antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (D) here, and in some other samples, appears to be somewhat better in the retention of immunoreactivity.
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f6: Retinal labeling with anti-vimentin antibody. All of the freeze substituted samples (A-E) show a higher level of immunoreactivity than the formalin-fixed sample (F). Of all the antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (D) here, and in some other samples, appears to be somewhat better in the retention of immunoreactivity.

Mentions: Figure 6, Figure 7, and Figure 8 show the results of labeling with antibodies to vimentin, a Type III intermediate filament protein, in the retina (Figure 6), cornea (Figure 7) and lens (Figure 8). Though vimentin is a common IF protein, it is not present in all cell types (e.g., the corneal epithelium lacks vimentin, while corneal keratocytes express it), providing a good internal control for non-specificity. Each of the freeze substitution variants (Figure 6A-E, Figure 7A-E, 8 and Figure 8A-E) was compared to formalin-preserved tissue (Figure 6F, Figure 7F, and Figure 8F). All of the freeze-substituted samples showed a greater preservation of immunoreactivity than formalin-fixed samples. Of all antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (Figure 6D), here and in some other samples, appears somewhat better in retention of immunoreactivity. In the cornea (Figure 7) there appears to be slightly better retention of reactivity in the corneal endothelium in Figure 7A and again in Figure 7D compared to other routines. In sections of the lens (Figure 8), there was more variability between different routines than was seen in the retina and cornea, but again, all freeze-substituted variants (Figure 8A-E) retained better immunoreactivity than formalin-fixed tissue. As in Figure 6 and Figure 7, the M-AA 48 RT appears strongest. The M-AA 1W appears to be the least reactive of the freeze-substituted tissues using the vimentin antibodies, but is still an improvement over formalin.


An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies.

Sun N, Shibata B, Hess JF, FitzGerald PG - Mol. Vis. (2015)

Retinal labeling with anti-vimentin antibody. All of the freeze substituted samples (A-E) show a higher level of immunoreactivity than the formalin-fixed sample (F). Of all the antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (D) here, and in some other samples, appears to be somewhat better in the retention of immunoreactivity.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403009&req=5

f6: Retinal labeling with anti-vimentin antibody. All of the freeze substituted samples (A-E) show a higher level of immunoreactivity than the formalin-fixed sample (F). Of all the antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (D) here, and in some other samples, appears to be somewhat better in the retention of immunoreactivity.
Mentions: Figure 6, Figure 7, and Figure 8 show the results of labeling with antibodies to vimentin, a Type III intermediate filament protein, in the retina (Figure 6), cornea (Figure 7) and lens (Figure 8). Though vimentin is a common IF protein, it is not present in all cell types (e.g., the corneal epithelium lacks vimentin, while corneal keratocytes express it), providing a good internal control for non-specificity. Each of the freeze substitution variants (Figure 6A-E, Figure 7A-E, 8 and Figure 8A-E) was compared to formalin-preserved tissue (Figure 6F, Figure 7F, and Figure 8F). All of the freeze-substituted samples showed a greater preservation of immunoreactivity than formalin-fixed samples. Of all antibodies tested, however, the disparity between freeze-substituted and formalin-fixed tissues was less with anti-vimentin labeling of the retina than in any other tissue or antibody we tested. The M-AA 48RT (Figure 6D), here and in some other samples, appears somewhat better in retention of immunoreactivity. In the cornea (Figure 7) there appears to be slightly better retention of reactivity in the corneal endothelium in Figure 7A and again in Figure 7D compared to other routines. In sections of the lens (Figure 8), there was more variability between different routines than was seen in the retina and cornea, but again, all freeze-substituted variants (Figure 8A-E) retained better immunoreactivity than formalin-fixed tissue. As in Figure 6 and Figure 7, the M-AA 48 RT appears strongest. The M-AA 1W appears to be the least reactive of the freeze-substituted tissues using the vimentin antibodies, but is still an improvement over formalin.

Bottom Line: Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment.The approach shows a notable improvement in preservation of immunoreactivity.On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Human Anatomy, School of Medicine, University of California Davis, Davis, CA.

ABSTRACT

Purpose: Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques.

Methods: A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed.

Results: An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ hybridization, lectin labeling, and the Click-iT chemistry approach to labeling the thymidine analog EdU. On the negative side, this approach dramatically reduced intrinsic GFP fluorescence.

Conclusions: A simple, cost-effective derivative of the freeze substitution process is described that is of particular value in the study of rodent or other small eyes, where fixation gradients, globe buckling, retinal detachment, differential shrinkage, autofluorescence, and tissue immunoreactivity have been problematic.

No MeSH data available.


Related in: MedlinePlus