Name: isolation of intact eyeball to obtain integral ocular surface tissue for histological examination and immunohistochemistry
Uploaded: 2019-10-20T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry at JoVE.com

The authors thank Prof. Rong Ju for critical reading of the manuscript, and all lab members for technical assistance. This work was supported by grants from the National Natural Science Foundation of China (NSFC: 31571077; Beijing, China), the Guangzhou City Sciences and Technologies Innovation Project (201707020009; Guangzhou, Guangdong Province, China), &#34;100 People Plan&#34; from Sun Yat-sen University (8300-18821104; Guangzhou, Guangdong Province, China), and research funding from State Key Laboratory of Ophthalmology at Zhongshan Ophthalmic Center (303060202400339; Guangzhou, Guangdong Province, China).

All procedures involving the use of mice were approved by the Animal Care and Use Committee, Zhongshan Ophthalmic Center, and adhered to ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Dissection of eyeball with intact ocular surface and eyelids
<ol>
	<li>Dissect the head.
	<ol>
		<li>Euthanize postnatal day 10 (P10) and P28 mice (see the Table of Materials for mouse strains) by cervical dislocation, cut the head off the neck by a pair of sharp sc...

<ol>
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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ocular+surface+development+and+gene+expression.">Ocular surface development and gene expression.</a> Journal of Ophthalmology. 2013, (2013).</li><li>Arita, R., Fukuoka, S., Morishige, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+Morphology+of+the+Lipid+Layer+of+the+Tear+Film.">Functional Morphology of the Lipid Layer of the Tear Film.</a> Cornea. 36 Suppl 1, S60-S66 (2017).</li><li>Guo, D., 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=Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene.">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2018).</li><li>Knop, E., Knop, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomy+and+immunology+of+the+ocular+surface.">Anatomy and immunology of the ocular surface.</a> Chemical Immunology and Allergy. 92, 36-49 (2007).</li><li>Mizoguchi, 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=Ocular+surface+alkali+injury+damages+meibomian+glands+in+mice.">Ocular surface alkali injury damages meibomian glands in mice.</a> The Ocular Surface. 15 (4), 713-722 (2017).</li><li>Gipson, I. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Goblet+cells+of+the+conjunctiva:+A+review+of+recent+findings.">Goblet cells of the conjunctiva: A review of recent findings.</a> Progress in Retinal and Eye Research. 54, 49-63 (2016).</li><li>Nowell, C. 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=Chronic+inflammation+imposes+aberrant+cell+fate+in+regenerating+epithelia+through+mechanotransduction.">Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction.</a> Nature Cell Biology. 18 (2), 168-180 (2016).</li><li>Nagata, M., Ohashi, Y., Ozawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+histochemical+study+of+the+development+of+premaxilla+and+maxilla+during+secondary+palate+formation+in+the+mouse+embryo.">A histochemical study of the development of premaxilla and maxilla during secondary palate formation in the mouse embryo.</a> Archives of Histology and Cytology. 54 (3), 267-278 (1991).</li><li>Zhang, 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=A+role+for+MEK+kinase+1+in+TGF-beta/activin-induced+epithelium+movement+and+embryonic+eyelid+closure.">A role for MEK kinase 1 in TGF-beta/activin-induced epithelium movement and embryonic eyelid closure.</a> The Embo Journal. 22 (17), 4443-4454 (2003).</li><li>Sharov, A. 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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=TGFbeta+signaling+inhibits+goblet+cell+differentiation+via+SPDEF+in+conjunctival+epithelium.">TGFbeta signaling inhibits goblet cell differentiation via SPDEF in conjunctival epithelium.</a> Development. 141 (23), 4628-4639 (2014).</li><li>Guo, D., 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=Ocular+surface+pathogenesis+associated+with+precocious+eyelid+opening+and+necrotic+autologous+tissue+in+mouse+with+disruption+of+Prickle+1+gene.">Ocular surface pathogenesis associated with precocious eyelid opening and necrotic autologous tissue in mouse with disruption of Prickle 1 gene.</a> Experimental Eye Research. 180, 208-225 (2019).</li></ol>

One critical reminder for preparation of the intact eyeball is that all orbital bones must be removed completely, especially the juga and lacrimal bones, which are small and located near the bottom of eye socket. Any left-over bones may complicate the ensuing histology. In case a tiny piece of bone was not completely removed from dissection by accident, it may be picked out from the embedding paraffin block using a pair of sharptweezers. The hole left behind should be filled with melted paraffin after this operation....

The ocular surface consists of a continuous sheet of regionalized epithelium including palpebral conjunctiva, bulbar conjunctiva and cornea1. Many glandular structures are associated with the ocular surface epithelium and together generate a layer of tear film protecting the cornea surface from drying and environmental invasions2. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). Both genetic factors and environmental irritants or their interactions contribute to pathological alterations of the OS3,4. Accordingly, a variety of genetically-engineered and physically or chemically-induced animal models have been used for studying disease processes of the human OS.
The structure and function of the mouse OS is similar to that of humans in many ways. The tear film components secreted by the ocular glands are also similar between mice and humans. A wealth of studies has been conducted using mouse models for elucidating mechanisms of human OS diseases5,6,7. In many occasions, it is critical to analyze global instead of local molecular changes of the OS to gain comprehensive information under the same experimental treatment. Therefore, sample preparation with good integrity is needed to ensure each part of the OS to be analyzed simultaneously.
The mouse OS tightly associates with the eyeball that was embedded in the eye socket/orbit (a bony cup made of several different skull bones) and connects to it through thin connective tissues. There exist tremendous challenges for dissecting the whole ocular surface without damaging the palpebral or bulbar conjunctiva. These challenges descend from: (i) the OS is soft and thin and affixed to physically separate yet movable eyelids and eyeball, therefore vulnerable for distortion and damage; and (ii) the limited space between the orbital bones and eyeball restrict the dissection operations. The challenges are much greater for the adult mouse. In the embryonic mouse, the orbital bone ossification is not complete and surrounding tissues are relative loose8. The head can be removed and bisected, and then directly subjected to paraformaldehyde (PFA) fixation and embedding9. By contrast, the postnatal and adult mouse orbital bones are fully ossified with thick surrounding tissues, making the penetration of fixatives less efficient. Furthermore, the orbital/skull bones are hard and brittle, easily broken when sectioning them in the soft embedding compounds such as paraffin. The broken pieces of bones will unanimously tear the nearby tissues resulting in inferior tissue morphology.
Many published studies often showed partial ocular surface, which may be sufficient for their particular research purposes10,11. A gross examination of literatures found only few studies showing the whole intact ocular surface being demonstrated without detailed description of dissection protocols12,13. In this protocol, we detailed a dissection method to obtain integral postnatal ocular surface, in which orbital and skull bones were orderly removed, leaving untouched ocular surface together with the eyeball and eyelids, minimizing the physical damages. We further examined the OS histology and performed immunohistochemistry using the tissue sections prepared with this protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1&#215; Phosphate buffered saline (PBS)</td>
			<td>Transgen Biotech</td>
			<td>FG701-01</td>
			<td></td>
		</tr>
		<tr>
			<td>50ml centrifuge tube</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive microscope slides</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12379</td>
			<td>Suggested concentration 1:500 - 1,000</td>
		</tr>
		<tr>
			<td>Alexa Fluor 568 Phalloidin</td>
			<td>Invitrogen/Life Technologies</td>
			<td>A12380</td>
			<td>Suggested concentration 1:500 - 1,000</td>
		</tr>
		<tr>
			<td>Anti-K12 antibody</td>
			<td>Abcam</td>
			<td>ab124975</td>
			<td>Suggested concentration 1:1,000</td>
		</tr>
		<tr>
			<td>Anti-K14 antibody</td>
			<td>Abcam</td>
			<td>ab7800</td>
			<td>Suggested concentration 1:800</td>
		</tr>
		<tr>
			<td>Citric acid</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cover slide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Curved forceps</td>
			<td>World Precision Instruments</td>
			<td>14127</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope.</td>
			<td>Olmpus</td>
			<td>SZ61</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethyl alcohol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent Microscope</td>
			<td>Zeiss</td>
			<td>AxioImager.Z2</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoromount-G Mounting media</td>
			<td>SouthernBiotech</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro dissecting scissors-straight blade</td>
			<td>World Precision Instruments</td>
			<td>503242</td>
			<td></td>
		</tr>
		<tr>
			<td>Microwave ovens</td>
			<td>Galanz</td>
			<td>P70D20TL-D4</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse strains</td>
			<td>C57/BL6 and Sv129 mixed</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>No.4 straight forceps</td>
			<td>World Precision Instruments</td>
			<td>501978-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde (PFA)</td>
			<td>Various</td>
			<td></td>
			<td>Prepare a 4% solution in 1&#215; PBS and filter with 0.45&#956;m filter membrane</td>
		</tr>
		<tr>
			<td>Tissue culture dish</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>SLBW6818</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation of intact eyeball to obtain integral ocular surface tissue for histological examination and immunohistochemistry

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. Disruption of OS would lead to keratitis, conjunctivitis or both (keratoconjunctivitis). In experimental animal models with certain genetic modifications or artificial operations, it is useful to examine all parts of the OS epithelial sheet to evaluate relative pathogenetic changes of each part in parallel. However, dissection of OS tissue as a whole without distortion or damage has been challenging, primarily due to the softness and thinness of the OS affixed to physically separate yet movable eyelids and eyeball. Additionally, the deep eye socket formed by the hard skull/orbital bones is fully occupied by the eyeball leaving limited space for operating dissections. As a result, direct dissection of the eyeball with associated OS tissues from the facial side would often lead to tissue damages, especially the palpebral and bulbar conjunctiva. In this protocol, we described a method to remove the skull and orbital bones sequentially from a bisected mouse head, leaving eyelids, ocular surface, lens and retina altogether in one piece. The integrity of the OS sheet was well preserved and could be examined by histology or immunostaining in a single section.

The major skull bones viewed from different perspectives were illustrated in Figure 1A-E, with colors denoting different bones. We used four-week old animal for demonstration of the dissection processes. Following dissection steps 1.1.1-1.1.3 and a short prefixation (step 1.1.4), the eyeball with associated facial bones are demonstrated in Figure 1E. Further trimming to remove anterior (nasal and...

Watch this Scientific Journal Video about Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry at JoVE.com

Isolation of Intact Eyeball to Obtain Integral Ocular Surface Tissue for Histological Examination and Immunohistochemistry

This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

Ocular surface (OS) consists of an epithelial sheet with three connected parts: palpebral conjunctiva, bulbar conjunctiva and corneal epithelium. ...

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