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Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

Preparation and Immunofluorescence Staining of Bundles and Single Fiber Cells from the Cortex and Nucleus of the Eye Lens

Published: June 9th, 2023

DOI:

10.3791/65638

1School of Optometry and Vision Science Program, Indiana University

This protocol describes methods to prepare peripheral, mature, and nuclear eye lens fiber cells for immunofluorescence staining to study complex cell-to-cell interdigitations and the membrane architecture.

The lens is a transparent and ellipsoid organ in the anterior chamber of the eye that changes shape to finely focus light onto the retina to form a clear image. The bulk of this tissue comprises specialized, differentiated fiber cells that have a hexagonal cross section and extend from the anterior to the posterior poles of the lens. These long and skinny cells are tightly opposed to neighboring cells and have complex interdigitations along the length of the cell. The specialized interlocking structures are required for normal biomechanical properties of the lens and have been extensively described using electron microscopy techniques. This protocol demonstrates the first method to preserve and immunostain singular as well as bundles of mouse lens fiber cells to allow the detailed localization of proteins within these complexly shaped cells. The representative data show staining of the peripheral, differentiating, mature, and nuclear fiber cells across all regions of the lens. This method can potentially be used on fiber cells isolated from lenses of other species.

The lens is a clear and ovoid tissue in the anterior chamber of the eye that is made up of two cell types, epithelial and fiber cells1 (Figure 1). There is a monolayer of epithelial cells that covers the anterior hemisphere of the lens. Fiber cells are differentiated from epithelial cells and make up the bulk of the lens. The highly specialized fiber cells undergo an elongation, differentiation, and maturation programming, marked by distinct changes in cell membrane morphology from the lens periphery to the lens center2,3,4

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Mice have been cared for based on an animal protocol approved by the Institutional Animal Care and Use Committee at Indiana University Bloomington. The mice used to generate representative data were control (wild-type) animals in the C57BL6/J background, female, and 8-12 weeks old. Both male and female mice can be used for this experiment, since the sex of the mice is very unlikely to affect the experiment's outcome.

1. Lens dissection and decapsulation

  1. Euthani.......

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Lens fiber cells are prepared from the lens cortex (differentiating fibers and mature fibers) and the nucleus, and the cells are stained with phalloidin for F-actin and WGA for the cell membrane. A mixture of bundles of cells or single lens fibers (Figure 3) are observed and imaged. From the lens cortex, two types of cells (Figure 3A) are found. Differentiating fiber cells in the lens periphery are straight, with very small protrusions along their short sides. A.......

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This protocol has demonstrated the fixation, preservation, and immunostaining methods that faithfully preserve the 3D membrane morphology of bundles or singular lens fiber cells from various depths in the lens. The stained lens fibers are compared with SEM preparations that have long been used to study lens fiber cell morphology. The results show comparable membrane structures between both preparations. EM remains the gold standard for studying cell morphology, but immunolabeling is more challenging in SEM samples for lo.......

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This work was supported by grant R01 EY032056 (to CC) from the National Eye Institute. The authors thank Dr. Theresa Fassel and Kimberly Vanderpool at the Scripps Research Core Microscopy Facility for their assistance with the electron microscope images.

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Name Company Catalog Number Comments
100% Triton X-100 FisherScientific BP151-500
60mm plate FisherScientific FB0875713A
16% paraformaldehyde Electron Microscopy Sciences 15710
10X phosphate buffered saline ThermoFisher 70011-044
1X phosphate buffered saline ThermoFisher 14190136
48-well plate CytoOne CC7672-7548
Cover slips (22 x 40 mm) FisherScientific 12-553-467
Curved tweezers World Precision Instruments 501981
Dissection microscope Carl Zeiss Stereo Discovery V8
Fine tip straight tweezers Electron Microscopy Sciences 72707-01
Fisherbrand Superfrost Plus Microscope Slides FisherScientific 12-550-15
LSM 800 confocal microscope with Airyscan (63X) and Zen 3.5 Software Carl Zeiss
Nail polish
Normal donkey serum Jackson ImmunoResearch 017-000-121
Phalloidin (rhodamine) ThermoFisher R415
Primary antibody
Scalpel Feather Disposable, steril, No. 11 VWR 76241-186
Secondary antibody
Straight forceps World Precision Instruments 11252-40
Thermo Scientific Nunc MicroWell MiniTrays (dissection tray) FisherScientific 12-565-154
Ultra-fine scissors World Precision Instruments 501778
VECTASHIELD Antifade Mounting Medium with DAPI Vector Laboratories H-1200
Wheat germ agglutinin (fluorescein) Vector Laboratories FL-1021-5

  1. Lovicu, F. J., McAvoy, J. W. Growth factor regulation of lens development. Developmental Biology. 280 (1), 1-14 (2005).
  2. Kuszak, J., Alcala, J., Maisel, H. The surface morphology of embryonic and adult chick lens-fiber cells. The American Journal of Anatomy. 159 (4), 395-410 (1980).
  3. Kuszak, J. R. The ultrastructure of epithelial and fiber cells in the crystalline lens. International Review of Cytology. 163, 305-350 (1995).
  4. Kuszak, J. R., Macsai, M. S., Rae, J. L. Stereo scanning electron microscopy of the crystalline lens. Scanning Electron Microscopy. , 1415-1426 (1983).
  5. Lo, W. K., Harding, C. V. Square arrays and their role in ridge formation in human lens fibers. Journal of Ultrastructure Research. 86 (3), 228-245 (1984).
  6. Taylor, V. L., et al. Morphology of the normal human lens. Investigative Ophthalmology & Visual Science. 37 (7), 1396-1410 (1996).
  7. Vrensen, G. F. Aging of the human eye lens-a morphological point of view. Comparative Biochemistry and Physiology. Part A, Physiology. 111 (4), 519-532 (1995).
  8. Vrensen, G. F., Duindam, H. J. Maturation of fiber membranes in the human eye lens. Ultrastructural and Raman microspectroscopic observations. Ophthalmic Research. 27, 78-85 (1995).
  9. Willekens, B., Vrensen, G. The three-dimensional organization of lens fibers in the rabbit. A scanning electron microscopic reinvestigation. Albrecht von Graefe's Archive for Clinical and Experimental Opthalmology. 216 (4), 275-289 (1981).
  10. Willekens, B., Vrensen, G. The three-dimensional organization of lens fibers in the rhesus monkey. Graefe's Archive for Clinical and Experimental Ophthalmology. 219 (3), 112-120 (1982).
  11. Zhou, C. J., Lo, W. K. Association of clathrin, AP-2 adaptor and actin cytoskeleton with developing interlocking membrane domains of lens fibre cells. Experimental Eye Research. 77 (4), 423-432 (2003).
  12. Kuwabara, T. The maturation of the lens cell: a morphologic study. Experimental Eye Research. 20 (5), 427-443 (1975).
  13. Weeber, H. A., Eckert, G., Pechhold, W., vander Heijde, R. G. Stiffness gradient in the crystalline lens. Graefe's Archive for Clinical and Experimental Ophthalmology. 245 (9), 1357-1366 (2007).
  14. Weeber, H. A., et al. Dynamic mechanical properties of human lenses. Experimental Eye Research. 80 (3), 425-434 (2005).
  15. Weeber, H. A., vander Heijde, R. G. On the relationship between lens stiffness and accommodative amplitude. Experimental Eye Research. 85 (5), 602-607 (2007).
  16. Heys, K. R., Cram, S. L., Truscott, R. J. Massive increase in the stiffness of the human lens nucleus with age: the basis for presbyopia. Molecular Vision. 10, 956-963 (2004).
  17. Heys, K. R., Friedrich, M. G., Truscott, R. J. Presbyopia and heat: changes associated with aging of the human lens suggest a functional role for the small heat shock protein, alpha-crystallin, in maintaining lens flexibility. Aging Cell. 6 (6), 807-815 (2007).
  18. Glasser, A., Campbell, M. C. Biometric, optical and physical changes in the isolated human crystalline lens with age in relation to presbyopia. Vision Research. 39 (11), 1991-2015 (1999).
  19. Pierscionek, B. K. Age-related response of human lenses to stretching forces. Experimental Eye Research. 60 (3), 325-332 (1995).
  20. Biswas, S. K., Lee, J. E., Brako, L., Jiang, J. X., Lo, W. K. Gap junctions are selectively associated with interlocking ball-and-sockets but not protrusions in the lens. Molecular Vision. 16, 2328-2341 (2010).
  21. Lo, W. K., et al. Aquaporin-0 targets interlocking domains to control the integrity and transparency of the eye lens. Investigative Ophthalmology & Visual Science. 55 (3), 1202-1212 (2014).
  22. Cheng, C., et al. Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties. Journal of Cell Science. 131 (23), (2018).
  23. Cheng, C., et al. Tropomodulin 1 regulation of actin is required for the formation of large paddle protrusions between mature lens fiber cells. Investigative Ophthalmology & Visual Science. 57 (10), 4084-4099 (2016).
  24. Kuszak, J. R. The development of lens sutures. Progress in Retinal and Eye Research. 14 (2), 567-591 (1995).
  25. Bassnett, S., Costello, M. J. The cause and consequence of fiber cell compaction in the vertebrate lens. Experimental Eye Research. 156, 50-57 (2017).
  26. Biswas, S., Son, A., Yu, Q., Zhou, R., Lo, W. K. Breakdown of interlocking domains may contribute to formation of membranous globules and lens opacity in ephrin-A5(-/-) mice. Experimental Eye Research. 145, 130-139 (2016).
  27. Blankenship, T., Bradshaw, L., Shibata, B., Fitzgerald, P. Structural specializations emerging late in mouse lens fiber cell differentiation. Investigative Ophthalmology & Visual Science. 48 (7), 3269-3276 (2007).
  28. Cheng, C., et al. Age-related changes in eye lens biomechanics, morphology, refractive index and transparency. Aging. 11 (24), 12497-12531 (2019).
  29. Cheng, C., et al. EphA2 affects development of the eye lens nucleus and the gradient of refractive index. Investigative Ophthalmology & Visual Science. 63 (1), 2 (2022).
  30. Cheng, C., Gokhin, D. S., Nowak, R. B., Fowler, V. M. Sequential application of glass coverslips to assess the compressive stiffness of the mouse lens: strain and morphometric analyses. Journal of Visualized Experiments. (111), e53986 (2016).
  31. Forrester, J. V., Dick, A. D., McMenamin, P. G., Roberts, F., Pearlman, E., Saunders, W. B. Anatomy of the eye and orbit. The Eye (Fourth Edition). , 1 (2016).
  32. Cheng, C., Nowak, R. B., Fowler, V. M. The lens actin filament cytoskeleton: Diverse structures for complex functions. Experimental Eye Research. 156, 58-71 (2017).
  33. Goldberg, M. W., Fiserova, J. Immunogold labeling for scanning electron microscopy. Methods in Molecular Biology. 1474, 309-325 (2016).
  34. Goldberg, M. W. High-resolution scanning electron microscopy and immuno-gold labeling of the nuclear lamina and nuclear pore complex. Methods in Molecular Biology. 1411, 441-459 (2016).
  35. Hermann, R., Walther, P., Muller, M. Immunogold labeling in scanning electron microscopy. Histochemistry and Cell Biology. 106 (1), 31-39 (1996).
  36. Gokhin, D. S., et al. Tmod1 and CP49 synergize to control the fiber cell geometry, transparency, and mechanical stiffness of the mouse lens. PLoS One. 7 (11), e48734 (2012).

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