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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

Low magnification overviews of H&E-stained paraffin sections of mouse eyes. A: Immersion in 10% neutral buffered formalin for 24 h. B: Freeze substitution in acetone. C: Freeze substitution in 97% methanol 3% acetic acid (M-AA). D: Same as in C, but skipping the butane freezing step, and freezing instead by direct immersion in −80 M-AA. Formalin-fixed eyes consistently showed shrinkage and buckling, and splitting caused by shear (white arrow, lens Figure 1A) as tissue fixed and shrank. Retinal detachment was common as well (white arrow, A). Acetone fixation (B) was good for the cornea, but not for most other ocular structures. Panel C shows tissue preserved by freeze substitution in methanol, or methanol:acetic acid. This level of preservation was typical of all the different routines described in Materials and Methods, but only the M-AA −80 is shown. Panel D shows that good quality can be achieved without the butane step, though, as shown in Figure 3, Figure 4, and Figure 5, some loss of quality can be seen on closer inspection. Higher magnification views of the retina, cornea, and lens are shown from each of the different freeze substitution routines in subsequent images. With the exception of the not-uncommon freezing cracks (black arrow C), the overall relationships of ocular structures are consistently well preserved, and lacking in buckling and retinal detachment.
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f2: Low magnification overviews of H&E-stained paraffin sections of mouse eyes. A: Immersion in 10% neutral buffered formalin for 24 h. B: Freeze substitution in acetone. C: Freeze substitution in 97% methanol 3% acetic acid (M-AA). D: Same as in C, but skipping the butane freezing step, and freezing instead by direct immersion in −80 M-AA. Formalin-fixed eyes consistently showed shrinkage and buckling, and splitting caused by shear (white arrow, lens Figure 1A) as tissue fixed and shrank. Retinal detachment was common as well (white arrow, A). Acetone fixation (B) was good for the cornea, but not for most other ocular structures. Panel C shows tissue preserved by freeze substitution in methanol, or methanol:acetic acid. This level of preservation was typical of all the different routines described in Materials and Methods, but only the M-AA −80 is shown. Panel D shows that good quality can be achieved without the butane step, though, as shown in Figure 3, Figure 4, and Figure 5, some loss of quality can be seen on closer inspection. Higher magnification views of the retina, cornea, and lens are shown from each of the different freeze substitution routines in subsequent images. With the exception of the not-uncommon freezing cracks (black arrow C), the overall relationships of ocular structures are consistently well preserved, and lacking in buckling and retinal detachment.

Mentions: Figure 2A-D presents low-magnification overviews of mouse eyes fixed by immersion in 10% formalin for 24 h (Figure 2A), freeze substituted with acetone (Figure 2B), freeze substituted with methanol:acetic acid (Figure 2C), and freeze substituted by direct immersion into −80 M-AA (no butane, Figure 2D). The formalin-fixed sample (Figure 2A) shows shrinkage/distortion in all ocular tissues, as well as areas of tissue splitting and retinal detachment (white arrows, Figure 2A). Acetone freeze substitution (Figure 2B) was unacceptable for most of the eye, but the cornea was well preserved.


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)

Low magnification overviews of H&E-stained paraffin sections of mouse eyes. A: Immersion in 10% neutral buffered formalin for 24 h. B: Freeze substitution in acetone. C: Freeze substitution in 97% methanol 3% acetic acid (M-AA). D: Same as in C, but skipping the butane freezing step, and freezing instead by direct immersion in −80 M-AA. Formalin-fixed eyes consistently showed shrinkage and buckling, and splitting caused by shear (white arrow, lens Figure 1A) as tissue fixed and shrank. Retinal detachment was common as well (white arrow, A). Acetone fixation (B) was good for the cornea, but not for most other ocular structures. Panel C shows tissue preserved by freeze substitution in methanol, or methanol:acetic acid. This level of preservation was typical of all the different routines described in Materials and Methods, but only the M-AA −80 is shown. Panel D shows that good quality can be achieved without the butane step, though, as shown in Figure 3, Figure 4, and Figure 5, some loss of quality can be seen on closer inspection. Higher magnification views of the retina, cornea, and lens are shown from each of the different freeze substitution routines in subsequent images. With the exception of the not-uncommon freezing cracks (black arrow C), the overall relationships of ocular structures are consistently well preserved, and lacking in buckling and retinal detachment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Low magnification overviews of H&E-stained paraffin sections of mouse eyes. A: Immersion in 10% neutral buffered formalin for 24 h. B: Freeze substitution in acetone. C: Freeze substitution in 97% methanol 3% acetic acid (M-AA). D: Same as in C, but skipping the butane freezing step, and freezing instead by direct immersion in −80 M-AA. Formalin-fixed eyes consistently showed shrinkage and buckling, and splitting caused by shear (white arrow, lens Figure 1A) as tissue fixed and shrank. Retinal detachment was common as well (white arrow, A). Acetone fixation (B) was good for the cornea, but not for most other ocular structures. Panel C shows tissue preserved by freeze substitution in methanol, or methanol:acetic acid. This level of preservation was typical of all the different routines described in Materials and Methods, but only the M-AA −80 is shown. Panel D shows that good quality can be achieved without the butane step, though, as shown in Figure 3, Figure 4, and Figure 5, some loss of quality can be seen on closer inspection. Higher magnification views of the retina, cornea, and lens are shown from each of the different freeze substitution routines in subsequent images. With the exception of the not-uncommon freezing cracks (black arrow C), the overall relationships of ocular structures are consistently well preserved, and lacking in buckling and retinal detachment.
Mentions: Figure 2A-D presents low-magnification overviews of mouse eyes fixed by immersion in 10% formalin for 24 h (Figure 2A), freeze substituted with acetone (Figure 2B), freeze substituted with methanol:acetic acid (Figure 2C), and freeze substituted by direct immersion into −80 M-AA (no butane, Figure 2D). The formalin-fixed sample (Figure 2A) shows shrinkage/distortion in all ocular tissues, as well as areas of tissue splitting and retinal detachment (white arrows, Figure 2A). Acetone freeze substitution (Figure 2B) was unacceptable for most of the eye, but the cornea was well preserved.

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