Name: using a laminating technique to perform confocal microscopy of the human sclera
Uploaded: 2016-05-06T14:00:00
Description: Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

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
<ol>
	<li>Prepa...

<ol>
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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...

Watch this Scientific Journal Video about Using a Laminating Technique to Perform Confocal Microscopy of the Human Sclera at JoVE.com

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 ...

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