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Visualizing lens epithelial cell proliferation in whole lenses.

Wiley LA, Shui YB, Beebe DC - Mol. Vis. (2010)

Bottom Line: Stained cells were readily localized with reference anatomic landmarks, like the transition zone.Double-labeling permitted the co-localization of markers in cycling cells.EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University, Saint Louis, MO 63110, USA. wileyl@vision.wustl.edu

ABSTRACT

Purpose: To develop a means to image cells in S-phase of the cell cycle while preserving the anatomic relationships within the lens.

Methods: Mice were injected with the thymidine analog, EdU. Whole lenses were removed, fixed and permeabilized. Cells that had incorporated EdU into their DNA were chemically labeled using fluorescent azides and "click" chemistry. Double labeling was performed with antibodies to other antigens, like phospho-histoneH3, a marker of mitotic cells. The position of labeled cells and lens anatomy was viewed using a simple device to position and flatten the lens.

Results: The nuclei of cells in S-phase of the cell cycle were intensely stained without the use of antibodies. Stained cells were readily localized with reference anatomic landmarks, like the transition zone. Whole lenses could be assayed by rotating the lens on the microscope stage. Double-labeling permitted the co-localization of markers in cycling cells.

Conclusions: EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens.

Show MeSH
Cell cycle detection and the proliferative landscape of whole lenses. A: EdU-positive (S-phase) cells in the whole lens from an 8-month-old lens. B: Cells undergoing mitosis are labeled using a fluorescent-conjugated antibody to phosphorylated-histoneH3 in an 8-month old lens. C: Total cells in an image field labeled with DRAQ-5. D: Merged image of all three labels detecting two phases of the cell cycle in the germinative zone of a whole lens. E: Whole lenses were placed anterior side down. Image stacks (275 μm) were acquired in the Z-plane from the center of the anterior pole of the lens, demarcated by the asterisk. Z-stacks were then projected in the Y-plane and joined together to re-create the entire lens image. Depth coding was then applied to each stack to provide relevant distance from the initiation point of each z-stack. Cooler colors are closer to the asterisk (origin of the z-stack) than are warmer colors. Using this method, the germinative zone of an entire lens can be seen in a 1-month old lens. F: Reconstruction of an 8-month-old lens showing the decrease in the number of cells in S-phase with age. G: A larger image of the boxed area in (E), showing the color scheme of the depth coding application. H: Larger image of the boxed area in (F). Scale bars: 10 μm (A-D) and 200 μm (E-H).
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f3: Cell cycle detection and the proliferative landscape of whole lenses. A: EdU-positive (S-phase) cells in the whole lens from an 8-month-old lens. B: Cells undergoing mitosis are labeled using a fluorescent-conjugated antibody to phosphorylated-histoneH3 in an 8-month old lens. C: Total cells in an image field labeled with DRAQ-5. D: Merged image of all three labels detecting two phases of the cell cycle in the germinative zone of a whole lens. E: Whole lenses were placed anterior side down. Image stacks (275 μm) were acquired in the Z-plane from the center of the anterior pole of the lens, demarcated by the asterisk. Z-stacks were then projected in the Y-plane and joined together to re-create the entire lens image. Depth coding was then applied to each stack to provide relevant distance from the initiation point of each z-stack. Cooler colors are closer to the asterisk (origin of the z-stack) than are warmer colors. Using this method, the germinative zone of an entire lens can be seen in a 1-month old lens. F: Reconstruction of an 8-month-old lens showing the decrease in the number of cells in S-phase with age. G: A larger image of the boxed area in (E), showing the color scheme of the depth coding application. H: Larger image of the boxed area in (F). Scale bars: 10 μm (A-D) and 200 μm (E-H).

Mentions: We performed immunostaining in adult lenses using a fluorescently-conjugated primary antibody against phosphorylated-histoneH3 (p-hH3), a marker for the mitotic phase of the cell cycle [9]. Double-staining of lenses with p-hH3 and EdU permits simultaneous observation of epithelial cells in the S- and M-phase of the cell cycle (Figure 3A-D). This approach should be applicable to other primary antibodies.


Visualizing lens epithelial cell proliferation in whole lenses.

Wiley LA, Shui YB, Beebe DC - Mol. Vis. (2010)

Cell cycle detection and the proliferative landscape of whole lenses. A: EdU-positive (S-phase) cells in the whole lens from an 8-month-old lens. B: Cells undergoing mitosis are labeled using a fluorescent-conjugated antibody to phosphorylated-histoneH3 in an 8-month old lens. C: Total cells in an image field labeled with DRAQ-5. D: Merged image of all three labels detecting two phases of the cell cycle in the germinative zone of a whole lens. E: Whole lenses were placed anterior side down. Image stacks (275 μm) were acquired in the Z-plane from the center of the anterior pole of the lens, demarcated by the asterisk. Z-stacks were then projected in the Y-plane and joined together to re-create the entire lens image. Depth coding was then applied to each stack to provide relevant distance from the initiation point of each z-stack. Cooler colors are closer to the asterisk (origin of the z-stack) than are warmer colors. Using this method, the germinative zone of an entire lens can be seen in a 1-month old lens. F: Reconstruction of an 8-month-old lens showing the decrease in the number of cells in S-phase with age. G: A larger image of the boxed area in (E), showing the color scheme of the depth coding application. H: Larger image of the boxed area in (F). Scale bars: 10 μm (A-D) and 200 μm (E-H).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2903465&req=5

f3: Cell cycle detection and the proliferative landscape of whole lenses. A: EdU-positive (S-phase) cells in the whole lens from an 8-month-old lens. B: Cells undergoing mitosis are labeled using a fluorescent-conjugated antibody to phosphorylated-histoneH3 in an 8-month old lens. C: Total cells in an image field labeled with DRAQ-5. D: Merged image of all three labels detecting two phases of the cell cycle in the germinative zone of a whole lens. E: Whole lenses were placed anterior side down. Image stacks (275 μm) were acquired in the Z-plane from the center of the anterior pole of the lens, demarcated by the asterisk. Z-stacks were then projected in the Y-plane and joined together to re-create the entire lens image. Depth coding was then applied to each stack to provide relevant distance from the initiation point of each z-stack. Cooler colors are closer to the asterisk (origin of the z-stack) than are warmer colors. Using this method, the germinative zone of an entire lens can be seen in a 1-month old lens. F: Reconstruction of an 8-month-old lens showing the decrease in the number of cells in S-phase with age. G: A larger image of the boxed area in (E), showing the color scheme of the depth coding application. H: Larger image of the boxed area in (F). Scale bars: 10 μm (A-D) and 200 μm (E-H).
Mentions: We performed immunostaining in adult lenses using a fluorescently-conjugated primary antibody against phosphorylated-histoneH3 (p-hH3), a marker for the mitotic phase of the cell cycle [9]. Double-staining of lenses with p-hH3 and EdU permits simultaneous observation of epithelial cells in the S- and M-phase of the cell cycle (Figure 3A-D). This approach should be applicable to other primary antibodies.

Bottom Line: Stained cells were readily localized with reference anatomic landmarks, like the transition zone.Double-labeling permitted the co-localization of markers in cycling cells.EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens.

View Article: PubMed Central - PubMed

Affiliation: Department of Ophthalmology and Visual Sciences, Washington University, Saint Louis, MO 63110, USA. wileyl@vision.wustl.edu

ABSTRACT

Purpose: To develop a means to image cells in S-phase of the cell cycle while preserving the anatomic relationships within the lens.

Methods: Mice were injected with the thymidine analog, EdU. Whole lenses were removed, fixed and permeabilized. Cells that had incorporated EdU into their DNA were chemically labeled using fluorescent azides and "click" chemistry. Double labeling was performed with antibodies to other antigens, like phospho-histoneH3, a marker of mitotic cells. The position of labeled cells and lens anatomy was viewed using a simple device to position and flatten the lens.

Results: The nuclei of cells in S-phase of the cell cycle were intensely stained without the use of antibodies. Stained cells were readily localized with reference anatomic landmarks, like the transition zone. Whole lenses could be assayed by rotating the lens on the microscope stage. Double-labeling permitted the co-localization of markers in cycling cells.

Conclusions: EdU labeling of whole lenses provides a simple, rapid and sensitive means to analyze lens epithelial cell proliferation in the anatomic context of the whole lens.

Show MeSH