Limits...
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

Photoreceptor outer segment autofluorescence. Formalin-fixed tissue exhibits a broad autofluorescence in both the green (A) and red (B) channels in the outer segments (os). Preservation by freeze substitution eliminated this autofluorescence (C-D).
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f11: Photoreceptor outer segment autofluorescence. Formalin-fixed tissue exhibits a broad autofluorescence in both the green (A) and red (B) channels in the outer segments (os). Preservation by freeze substitution eliminated this autofluorescence (C-D).

Mentions: Formalin-fixed retinas exhibit a broad-spectrum autofluorescence in the photoreceptor outer segments (os). This can be seen in Figure 11A-B which show fluorescence in both green and red channels. The use of freeze substitution essentially eliminates this, as can be seen in Figure 11C-D, which were exposed for the same time as Figure 11A-B in both red and green channels.


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)

Photoreceptor outer segment autofluorescence. Formalin-fixed tissue exhibits a broad autofluorescence in both the green (A) and red (B) channels in the outer segments (os). Preservation by freeze substitution eliminated this autofluorescence (C-D).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f11: Photoreceptor outer segment autofluorescence. Formalin-fixed tissue exhibits a broad autofluorescence in both the green (A) and red (B) channels in the outer segments (os). Preservation by freeze substitution eliminated this autofluorescence (C-D).
Mentions: Formalin-fixed retinas exhibit a broad-spectrum autofluorescence in the photoreceptor outer segments (os). This can be seen in Figure 11A-B which show fluorescence in both green and red channels. The use of freeze substitution essentially eliminates this, as can be seen in Figure 11C-D, which were exposed for the same time as Figure 11A-B in both red and green channels.

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