German Research Foundation (FOR2240 "(Lymph) Angiogenesis and Cellular Immunity in Inflammatory Diseases of the Eye" to CC and LMH; HE 6743/2-1 and HE 7643/3-1 to LMH; CU47/6-1 to CC), German Cancer Aid (to LMH and CC), GEROK program University of Cologne (to SLS and LMH), and EU COST BM1302 "Joining Forces to Corneal Regeneration" (to CC).

The use of human tissues must be reviewed and approved by an institutional review board or the equivalent. The work described here was approved by the local ethics committee and had approval for scientific examination. This work was performed according to the Declaration of Helsinki. The human scleral specimens were obtained from the eyes of globe donors (maximum post-mortem time 24 hr) at the Eye Bank of the Department of Ophthalmology, University of Cologne, Germany.
1. Experimental Preparation
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	<li>Prepa...

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The authors declare no financial disclosures. The contents of this article are solely the responsibility of the authors.

Laminating the human sclera is a method for performing confocal microscopy on this tissue. A critical step in this process is the use of ethanol instead of formalin for affixing the tissue. In our experience, better results are obtained when using ethanol instead of formalin for fixation. Blunt scalpels aggravate the procedure and should be avoided. Similarly, the drying up of the sclera should be avoided, as it complicates the procedure and reduces the quality of the images.
Regarding the imm...

The sclera is the rigid outer layer that covers the eye, which is made of dense connective tissue. It helps to protect intraocular structures and to maintain intraocular pressure. Thus, the sclera is essential for clear vision. It is devoid of lymphatic vessels 1,2 and thereby forms an outer lymphatic-free border between it and the lymphatic-free inner eye 3-7. It also provides attachment sites for extraocular muscles, thereby sharing anatomical similarities with tendons. Because the sclera mainly consists of dense bundles of type I collagen and has smaller numbers of collagen types III, IV, V, VI, VIII 8,9 and elastin 10,11, this tissue is not easy to use for immunohistochemistry.
Anatomically, the sclera can be separated into three main layers: (1) the superficial vascularized episclera, found underneath the conjunctiva and tenon&#39;s capsule and towards the sides and the back of the eye facing the orbit; (2) the scleral stroma, the main part of the sclera; and (3) the lamina fusca, which is a thin, pigmented layer located directly above the uvea. Our anatomical knowledge about the sclera stems mainly from the first half of the 20th century. At that time, researchers studied the anatomy of vasculature mainly by using India ink injections 12 and vascular casting 13-15. Later, it was researched in angiographic studies 16-19.
Since that time, older techniques have been improved and new ones have been developed that have allowed us to supplement previous anatomical knowledge. For example, it has only been about one decade since we have had such reliable lymphatic markers as lymphatic vascular endothelium specific hyaluronan receptor-1 (LYVE1) 20 or podoplanin 21. Confocal microscopy offers new possibilities for studying the anatomical features of the different tissues of the eye. It allows for multiple stains to be used for differentiating markers of cells or for the localization of cells in relation to blood vessels and other anatomical structures. It provides an overview when the sample is of a larger size and allows us to scan through a sample when in search of a specific cell type. With Z-Stack technology, confocal microscopy can be used for samples up to 100-200 &#181;m. The sclera differs in thickness between 0.3 mm behind the muscle insertions and 1 mm at the posterior pole 11. Due to both its thickness and opaqueness, the sclera is not suitable for confocal microscopy using traditional methods.
To remedy this, scleral tissues were laminated to allow for their analysis with confocal microscopy. This technology is useful for gaining a better understanding of both physiological and pathological situations in the human sclera.

<table>
	<tbody>
		<tr>
			<td>96% ethanol</td>
			<td>Merck Chemicals, Darmstadt, Germany</td>
			<td>P075.4</td>
			<td></td>
		</tr>
		<tr>
			<td>binocular stereo microscope&#160;</td>
			<td>Motic, Hongkong, China</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>26G needles&#160;</td>
			<td>Terumo, Leuven, Belgium</td>
			<td>303800</td>
			<td></td>
		</tr>
		<tr>
			<td>15.5mm trepan</td>
			<td>Geuder, Heidelberg, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
		<tr>
			<td>no.10 scalpel&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td>2E+08</td>
			<td></td>
		</tr>
		<tr>
			<td>ophthalmic scalpel micro feather&#160;</td>
			<td>Feather, pfm medical, Osaka, Japan</td>
			<td colspan="2">no. 7657BR</td>
		</tr>
		<tr>
			<td>CD 31 antibody (monoclonal mouse anti human)</td>
			<td>Dako, USA</td>
			<td>IR610</td>
			<td></td>
		</tr>
		<tr>
			<td>LYVE1 antibody&#160; (polyclonal rabbit anti human)</td>
			<td>Zytomed, Germany</td>
			<td colspan="2">RBK014-05</td>
		</tr>
		<tr>
			<td>goat anti mouse FITC antibody</td>
			<td>Sigma Aldrich, Steinheim, Germany</td>
			<td>F0257</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti rabbit Cy3 antibody</td>
			<td>Dianova, Germany</td>
			<td colspan="2">111-165-003</td>
		</tr>
		<tr>
			<td>Goat Serum normal</td>
			<td>Dako, Glostrup, Denmark</td>
			<td colspan="2">X090710-8</td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Carl Roth GmbH, Karlsruhe, Germany</td>
			<td>6335.1</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides&#160;</td>
			<td>Engelbrecht, Ederm&#252;nde, Germany</td>
			<td colspan="2">WC7695002</td>
		</tr>
		<tr>
			<td>Coverslips 24x24mm</td>
			<td>Th. Gayer, Lohmar, Germany</td>
			<td>7695026</td>
			<td></td>
		</tr>
		<tr>
			<td>DAKO fluorescent mounting medium&#160;</td>
			<td>DAKO, USA</td>
			<td>S3023</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM Meta 510 confocal microscopy&#160;</td>
			<td>Carl Zeiss AG, Jena, Germany</td>
			<td>n.a</td>
			<td></td>
		</tr>
	</tbody>
</table>

using a laminating technique to perform confocal microscopy of the human sclera

The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and VII). Due to its autofluorescence, opaqueness, and thickness, it has not been found suitable for confocal microscopy. An alternative approach to the one presented here, which uses formalin-fixed sclera embedded in paraffin for immunohistochemistry, has technical challenges, especially when preheating the tissue for antigen retrieval. Since the sclera is relatively poor in both cells and vessels, the use of larger tissue samples was explored to help prevent overlooking cells and to understand their localization in relation to vessels and other anatomical sites. To allow for the analysis of larger tissue samples under the confocal microscope, a laminating technique was performed to create thin layers from the sclera. Following the analysis of results of CD31 blood vessels and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1) positive cells, for which approval for scientific examination was obtained, the advantages and limitations of this method are discussed.

In the representative experiments performed here, there are demonstrable benefits derived from the use of this particular laminating technique. The first experiment illustrates the diverse network of the episcleral blood vessel plexus in three representative pictures (Figure 3). The vessels are positive for CD31.
The second experiment shows immune cells, in particular LYVE1 + cells of the episclera and their rel...

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Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera

Human sclera tissue is mainly collagen; therefore, it is not easily usable for immunohistochemistry. To achieve the goal of performing immunohistochemistry for confocal microscopy of scleral tissue, a laminating technique was used.


The sclera is a dense connective tissue that covers and protects the eye. It mainly consists of dense collagen bundles (types I, III, IV, V, VI, and ...

The overall goal of this methodology, is to perform confocal microscopy of the human sclera. This method can help to answer key questions in the ophthalmic field. Such as immune cell distribution and vascular status in the healthy, and the pathological conditions in the Sclera.The main advantage of this technique is that it allows for the confocal imaging of dense connective tissue. Demonstrating the procedure will be Stefan Kremers, a grad student from our laboratory. To begin, prepare 96 percent Ethanol and PBS in individual tubes.Then prepare 100 micro-liters per sample of the primary antibodies in the recommended dilution of PBS containing two percent BSA, and keep all solutions on ice. Arrange clean instruments, including one to two Colibri forceps, straight micro-dissecting scissors, curved scissors, a number 10 scalpel, or ophthalmic scalpel micro feather, four 26 gauge needles, and a swab. Wrap aluminum foil around a polystyrene plate, or prepare a cork plate to affix the tissue.After producing an anteriorly opened bulb according to the text protocol, place the Sclera on a swab with the open part facing up. Then use colibri forceps to remove the retina, and the pigmented uveal layers from the inner sclera until the sclera is free of these layers. Next use curved scissors if necessary to remove the retina and uvea from the papilla.Then remove the remaining conjunctiva, extra-ovular muscles, and tenons capsule from the superficial sclera. Now using colibri forceps to gently hold the tissue, with straight scissors cut approximately two square centimeter sized scleral samples from different locations on the tissue, avoiding repeated grabbing with the forceps which will make the area unsuitable for confocal microscopy. Place the samples in 1.5 milliliter tubes, add 1.5 milliliters of 96 percent ethanol, and incubate for 15 minutes to fix the tissue.Then after removing the ethanol, use 1.5 milliliters of PBS to wash the samples three times for five minutes each while shaking. Next, transfer the samples to 1.5 milliliters of PBS with five percent BSA, and incubate at room temperature for two hours which will induce swelling of the samples, to help with laminating and prevent nonspecific binding. When the samples have swelled, under a stereo-binocular microscope use 26 gauge needles that have been bent at the edges to affix the posterior scleral samples to the foil wrapped polystyrene membrane, or cork plate.Then to laminate the full thickness samples use colibri forceps to horizontally hold the anterior edge of the scleral tissue. With a number 10 scalpel, carefully cut as thin a layer as possible of the superficial sclera, from the underlying layer. To prevent the tissue from drying out pipette 50 micro-liters of PBS onto the layers.Then after the layers have been cut place them in 100 micro-liters of PBS in a 96 well plate, and use a waterproof marker to label both the orientation and the layer. Continue to isolate layers and transfer to the 96 well plate until the tissue is fully laminated. To carry out Immunohistochemistry, remove the medium from the 96 well plate by holding the pipette against the wall of each well and aspirating the fluid.Then add 100 micro-liters of the primary antibodies diluted one to 100 in PBS with two percent BSA to the appropriate wells, and incubate at four degrees Celsius overnight. The next day use 200 to 300 micro-liters of PBS to wash the samples on a shaker three times for five minutes each. If needed, use another primary antibody and incubate again overnight at four degrees Celsius.Then add 100 micro-liters of the corresponding secondary antibodies, diluted one to 300 in PBS with two percent goat serum, and incubate in the dark at room temperature for one to two hours. After the incubation, use 200 to 300 micro-liters of PBS to rinse the samples again on the shaker three times for five minutes each. Then add 100 micro-liters of a one to 2000 dilution of DAPI to each well and incubate in the dark at room temperature.Before using 200 to 300 micro-liters of PBS to wash the samples two times. Transfer the samples onto microscope slides under the stereo-binocular microscope to make sure the sample is lying flat on the slide, and add one to two drops of fluorescent mounting medium. Then place a cover slip on top and use transparent nail polish to seal the edges.Store in the dark at four degrees Celsius for later use or examine directly by confocal microscopy. To verify the thickness of the samples take z-stacks as the thickness differs equatorially and posteriorly depending on the scleral location. These images show the diverse network of the blood vessel plexis in laminated human sclera derived from the anterior and posterior episclera.The blood vessels are positive for CD31 as shown in green. In this figure, LYVE1 positive immune cells of the episclera and their relationship to the CD31 positive blood vessels are illustrated. The z-stack series was taken at 10x magnification to examine the three dimensional relationships of blood vessels and immune cells.As shown here the extra-ocular muscles enclosed within the sclera can also be analysed for the presence of blood vessels or immune cells, using the markers CD31 and LYVE1 respectively. Once mastered the laminating technique can be done in less than one hour if it is performed properly. While attempting this procedure it is important to remember to prevent the tissue from drying out.Don't forget that working with human tissue can be potentially infectious and precautions such as microbiological and virological diagnostics should always be taken beforehand.

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7.5K visualizações.  University of Cologne. The overall goal of this methodology, is to perform confocal microscopy of the human sclera. This method can help to answer key questions in the ophthalmic field. Such as immune cell distribution and vascular status in the healthy, and the pathological conditions in the Sclera.The main advantage of this technique is that it allows for the confocal imaging of dense connective tissue. Demonstrating the procedure will be Stefan Kremers, a grad student from our laboratory. To begin, prepar...