Name: Author Spotlight: Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining
Uploaded: 2023-06-09T14:50:00
Description: Watch this Scientific Journal Video about Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining at JoVE.com

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.

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&#39;s outcome.
1. Lens dissection and decapsulation
<ol>
	<li>Euthani...

Immunofluorescence Staining of Membrane Interdigitations in Lens Fiber Cells

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

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...

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 center2,3,4,5,6,7,8,9,10,11,12, also known as the lens nucleus. The function of the lens to fine-focus light coming from various distances onto the retina depends on its biomechanical properties, including stiffness and elasticity13,14,15,16,17,18,19. The complex interdigitations of lens fibers have been hypothesized20,21 and recently shown to be important for lens stiffness22,23.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/65638/65638fig01.jpg" /> 
Figure 1: Lens anatomy diagrams and representative scanning electron microscopy (SEM) images from lens fibers. The cartoon shows a longitudinal (anterior to posterior from top to bottom) view of the anterior monolayer of epithelial cells (shaded in light blue) and a bulk mass of lens fiber cells (white). The center of the lens (shaded in pink) is known as the nucleus and comprises highly compacted fiber cells. On the right, a cross-section cartoon reveals the elongated hexagon cell shape of lens fibers that are packed into a honeycomb pattern. Fiber cells have two broad sides and four short sides. Representative SEM images along the bottom show the complex membrane interdigitations between lens fiber cells at different depths of the lens. From the right, newly formed lens fibers at the lens periphery have small protrusions along the short sides and balls-and-sockets along the broad side (red boxes). During maturation, lens fibers develop large paddle domains that are decorated by small protrusions along the short sides (blue boxes). Mature fiber cells possess large paddle domains illustrated by small protrusions. These interlocking domains are important for lens biomechanical properties. Fiber cells in the lens nucleus have fewer small protrusions along their short sides and have complex tongue-and-groove interdigitations (purple boxes). The broad sides of the cell display a globular membrane morphology. The cartoon was modified from22,32 and not drawn to scale. Scale bar = 3 &#181;m. <a href="https://www.jove.com/files/ftp_upload/65638/65638fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The lens grows by adding shells of new fiber cells overlaid on top of previous generations of fibers24,25. Fiber cells have an elongated, hexagonal cross section shape with two broad sides and four short sides. These cells extend from the anterior to the posterior pole of the lens, and depending on the species, the lens fibers can be several millimeters in length. To support the structure of these elongated and skinny cells, specialized interdigitations along the broad and short sides create interlocking structures to maintain the lens shape and biomechanical properties. Changes in cell membrane shape during fiber cell differentiation and maturation have been extensively documented by electron microscopy (EM) studies2,3,4,5,6,7,8,9,10,20,26,27,28,29. Newly formed fiber cells have balls-and-sockets along their broad sides with very small protrusions along their short sides, while mature fibers have interlocking protrusions and paddles along their short sides. Nuclear fibers display tongue-and-groove interdigitations and globular membrane morphology. Little is known about the proteins that are required for these complex interlocking membranes. Previous studies on protein localization in fiber cells have relied on lens tissue sections, which do not allow clear visualization of the complex cell architecture.
This work has created and perfected a novel method to fix single and bundles of lens fiber cells to preserve the complex morphology and to allow immunostaining for proteins at the cell membrane and within the cytoplasm. This method faithfully preserves cell membrane architecture, comparable to data from EM studies, and allows staining with primary antibodies for specific proteins. We have previously immunostained cortical lens fibers undergoing differentiation and maturation22,23. In this protocol, there is also a new method to stain fiber cells from the lens nucleus. This protocol opens the door to understanding the mechanisms for formation and changes in membrane interdigitations during fiber cell maturation and lens nucleus compaction.

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			<td >100% Triton X-100</td>
			<td >FisherScientific</td>
			<td >BP151-500</td>
			<td ></td>
		</tr>
		<tr >
			<td >60mm plate</td>
			<td >FisherScientific</td>
			<td >FB0875713A</td>
			<td></td>
		</tr>
		<tr >
			<td >16% paraformaldehyde</td>
			<td >Electron Microscopy Sciences</td>
			<td >15710</td>
			<td></td>
		</tr>
		<tr >
			<td >10X phosphate buffered saline</td>
			<td >ThermoFisher</td>
			<td >70011-044</td>
			<td></td>
		</tr>
		<tr >
			<td >1X phosphate buffered saline</td>
			<td >ThermoFisher</td>
			<td >14190136</td>
			<td></td>
		</tr>
		<tr >
			<td >48-well plate</td>
			<td >CytoOne</td>
			<td >CC7672-7548</td>
			<td></td>
		</tr>
		<tr >
			<td >Cover slips (22 x 40 mm)</td>
			<td >FisherScientific</td>
			<td >12-553-467</td>
			<td></td>
		</tr>
		<tr >
			<td >Curved tweezers</td>
			<td >World Precision Instruments</td>
			<td >501981</td>
			<td></td>
		</tr>
		<tr >
			<td >Dissection microscope</td>
			<td >Carl Zeiss</td>
			<td >Stereo Discovery V8</td>
			<td></td>
		</tr>
		<tr >
			<td >Fine tip straight tweezers</td>
			<td >Electron Microscopy Sciences</td>
			<td >72707-01</td>
			<td></td>
		</tr>
		<tr >
			<td >Fisherbrand Superfrost Plus Microscope Slides</td>
			<td >FisherScientific</td>
			<td >12-550-15</td>
			<td></td>
		</tr>
		<tr >
			<td >LSM 800 confocal microscope with Airyscan (63X) and Zen 3.5 Software</td>
			<td >Carl Zeiss</td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Nail polish</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Normal donkey serum</td>
			<td >Jackson ImmunoResearch</td>
			<td >017-000-121</td>
			<td></td>
		</tr>
		<tr >
			<td >Phalloidin (rhodamine)</td>
			<td >ThermoFisher</td>
			<td >R415</td>
			<td></td>
		</tr>
		<tr >
			<td >Primary antibody</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Scalpel Feather Disposable, steril, No. 11</td>
			<td >VWR</td>
			<td >76241-186</td>
			<td></td>
		</tr>
		<tr >
			<td >Secondary antibody</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Straight forceps</td>
			<td >World Precision Instruments</td>
			<td >11252-40</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermo Scientific&#160;Nunc MicroWell MiniTrays (dissection tray)</td>
			<td >FisherScientific</td>
			<td >12-565-154</td>
			<td></td>
		</tr>
		<tr >
			<td >Ultra-fine scissors</td>
			<td >World Precision Instruments</td>
			<td >501778</td>
			<td></td>
		</tr>
		<tr >
			<td >VECTASHIELD Antifade Mounting Medium with DAPI</td>
			<td >Vector Laboratories</td>
			<td >H-1200</td>
			<td></td>
		</tr>
		<tr >
			<td >Wheat germ agglutinin (fluorescein)</td>
			<td >Vector Laboratories</td>
			<td >FL-1021-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation and immunofluorescence staining of bundles and single fiber cells from the cortex and nucleus of the eye lens

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.

Author Spotlight: Advancing Lens Biomechanics Research Through a Novel Protocol for Imaging Complex Interdigitations and Protein Staining 

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

The lens is a transparent ellipsoid organ in the anterior chamber of the eye, and is responsible for fine focusing of light onto the retina to create a clear image. The function of the lens relies on its biomechanical properties, complex interdigitations between the lens fiber cells has recently been shown to be important for lens stiffness. While the specialized morphology of lens fiber cells has been previously described by electron microscopy, little is known about the proteins that are required for these interlocking membranes.Previous studies have relied on lens tissue sections, which do not allow clear visualization of complex cell architecture. We have created and perfected a novel technique to faithfully preserve the complex membrane interdigitations between lens fiber cells for staining and confocal microscopy imaging of specific proteins. In this protocol, there is also a new method for staining the fiber cells from the center of the lens, which is also known as the lens nucleus.This method opens the door to understanding fiber cell differentiation and maturation by studying the mechanisms of formation and changes in membrane interdigitations.

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 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 ...

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JoVE Journal

Developmental Biology

Mouse Lens Dissection and Processing to Study Complex Cell-to-Cell Interdigitations and the Membrane Architecture

Begin by positioning a euthanized mouse for eye enucleation using curved forceps to press the tissue around the eye to displace the eye out of the socket. Then lift and remove the eye, and transfer it to fresh PBS in the dissection tray. Using ultra fine scissors, cut the optic nerve as close as possible to the eyeball, and carefully insert fine tip straight tweezers into the eyeball through the optic nerve's exit at the posterior of the eye.Now, insert scissors at the posterior of the eye and begin making an incision from the posterior toward the corneal scleral junction, and continue the incision until half of the junction has been separated. Gently push on the cornea so that the lens can exit through the incision. Using fine tip straight tweezers, carefully remove any large pieces of tissue from the lens.After finding the equatorial region, shallowly pierce the lens, and then remove the lens capsule. Transfer the lens fiber cells to a 60 millimeter dish with 1%paraformaldehyde. Using a sharp scalpel, split the ball of the fiber cells in half along its anterior posterior axis.And then further cut the halves along the same axis to produce quarters. Use the straight tweezers to remove the nucleus region from the lens fiber cell quarter. Next, add 200 microliters of 1%paraformaldehyde to a 48-well plate.Transfer the lens cortex to the plate and incubate for 15 minutes at room temperature with gentle shaking at 300 RPM. After blocking, add appropriate primary antibodies to the 48-well plate and transfer samples to the primary antibody solution. Incubate the samples overnight at 4 degrees Celsius with gentle shaking or mutation.The next day, wash the samples with PTX, and similarly, incubate them with secondary antibodies. After washing the samples, add one drop or 50 microliters of mounting media onto a plus charged microscope slide. Place the tissue in the mounting media.And use tweezers to gently separate the fiber cells from each other. Then, gently place a cover slip on top of the separated cells and mounting media. After removing excess media, use nail polish to seal the edges of the cover slip on the slide before confocal microscopy.After the lens dissection and lens capsule removal, transfer the fiber cell mass to wet gloved fingertips and gently roll in all directions to separate the lens nucleus. Then, transfer the lens nucleus to freshly made 1%paraformaldehyde solution in a 48-well plate and incubate overnight at 4 degrees Celsius with gentle shaking or mutation. The next day, transfer the sample to a 60 millimeter dish with 1%paraformaldehyde and use a sharp scalpel to split the lens nucleus along the anterior posterior axis in half, then into quarters.Then, postfix, block, stain, and mount the sample as previously demonstrated In these preparations, bundles of lens fiber cells with unique morphologies are found from different lens regions. The staining of the F-actin network shows enrichment at the cell membrane in differentiating and mature fibers while F-actin signals are present in the cytoplasm of nuclear fibers. Scanning electron microscopy and confocal images of differentiating fiber cells show ball and socket interdigitations with small interlocking protrusions, whereas mature fibers have paddles decorated by small protrusions.Scanning electron microscopy and confocal microscope images of nuclear lens fiber cells reveal infrequent and larger interlocking protrusions on the short sides of the cells where the cell membrane is rough and has tongue and groove interdigitations in these cells from the center of the lens.