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 scissors.</li>
		<li>Bisect head with a pair of straight scissors along the sagittal midline beginning at the interparietal bone rostrally to the nasal (Figure 1A,B). 
		NOTE: The skull bone hardens with mouse aging, be careful to keep the scissors cutting along the midline as much as possible.</li>
		<li>Place each half of the bisected head in a clean Petri dish, cut off the jaw and tongue first using sharp scissors. Free the optic nerve by cutting it off the brain prior to the optic chiasm location, and remove all brain tissues including olfactory bulb attached to the skull wall.</li>
		<li>Now the remaining tissues shall have all skull/orbital bones associated with the eyeball (Figure 1C,D). Wash the remaining part with PBS to clean blood and hair debris.</li>
		<li>Prefix tissues with 4% paraformaldehyde (PFA, in phosphate-buffered saline [PBS], pH 7.4) in a 50 mL conical tube at room temperature (RT) with rotation. The approximate fixation time is as follows: ~10 min for P0 to P7; ~20 min for P8 to P28; and ~30 min for P29 and older. 
		NOTE: The prefixation offers tissue rigidity making the ensuing dissection easier. Additionally, prefixation avoids leaving fresh tissue unfixed too long before dissection is complete. 
		CAUTION: PFA is hazardous and must be handled with care.</li>
	</ol>
	</li>
	<li>Remove skull/orbital bones.
	<ol>
		<li>After prefixation, quickly wash the dissected head in PBS three times to further eliminate the broken hairs and fixatives in order to proceed with further dissection. 
		CAUTION: Exposure to fixatives during dissection can be injurious to health.</li>
		<li>Place the tissue in 10 cm Petri dish, cut the skull into three parts along the planes indicated in Figure 1D,E. The eyeball in the socket is hidden in the middle part of the skull under ethmoid, frontal and maxillary bones (Figure 1D,E).</li>
		<li>Use two pairs of No. 4 straight forceps to peel off the ethmoid bone to fully expose the frontal and maxillary bones (Figure 1F).</li>
		<li>Geographically divide the skull surface (including maxillary and the frontal bones) into 4 areas (Figure 1F). Insert the tip of curved forceps horizontally into each area to remove the maxillary and frontal bones sequentially (Figure 1F). 
		NOTE: The maxillary bones cannot all be removed at one time. Patiently dissect them piece by piece. The frontal bone is directly connected to the eyeball through soft connective tissues. Take caution when removing it from the eyeball to prevent stretching the eyeball and damaging the conjunctiva.</li>
		<li>After removal of partial maxillary bone and all frontal bone, the underlying lacrimal and jugal bones would be exposed (Figure 1G). Remove the two bones and associated subcutaneous muscles and fats surrounding the eyeball, trim the eyeball with attached eyelids and skin into a small square-shape block to reduce tissue volume and facilitate orienting the tissue when embedding (Figure 1H).</li>
		<li>Place the eyeball and associated tissues back into 4% PFA and continue to fix overnight at 4 &#176;C. The fixed tissue can be preserved for at least one month at 4 &#176;C in PBS with addition of 0.02% NaN3. Alternatively, proceed to histological analysis immediately. 
		NOTE: If the tissue needs to be stored for longer periods, it can readily be stored in 70% ethanol.</li>
	</ol>
	</li>
</ol>
2. Histological analysis
<ol>
	<li>Paraffin section and hematoxylin and eosin (H&#38;E) staining
	<ol>
		<li>Follow standard protocol described elsewhere9 to perform paraffin embedding and sectioning.</li>
	</ol>
	</li>
	<li>Immunohistochemistry (IHC)
	<ol>
		<li>Dewax the paraffin sections with xylene and rehydrate the sections through alcohol series (100%, 95%, 80%) into distilled water (dH2O).</li>
		<li>Perform antigen retrieval by microwave treatment of the tissue slides in 0.01 M citric acid buffer (3 g of trisodium citrate, 0.4 g citric acid per 1 L double distilled H2O) in a glass slide jar with low power (120 W). Energy should be intermittently delivered for total 5-10 min with each interval lasting about 2 min. 
		NOTE: High-power microwave or consistent heating would lead to detachment of tissue sections from the slide.</li>
		<li>Pick out the slides from glass jar, carefully wipe off residual liquid surrounding each section using facial tissues and place them onto the slide rack in a histology box. Draw a square around each section with a waterproof histological pen. Place 100 &#181;L of blocking buffer (0.1% triton X-100 and 10% donkey serum in 1x PBS) onto each square, and incubate for 30 min at RT.</li>
		<li>Carefully remove the blocking solution with vacuum, add the primary antibody (see the Table of Materials) with desired dilution in blocking buffer, and continue to incubate the slides at 4 &#176;C for 24 h or longer. 
		NOTE: The concentration for each primary antibody used for IHC varies, and needs to be tested out in pilot experiments.</li>
		<li>Wash tissue slides with PBST (0.1% Triton in PBS) three times, for 10 min each. Repeat step 2.2.4 using the secondary antibody (see Table of Materials) together with 4&#39;,6-diamidino-2-phenylindole (DAPI) replacing the blocking buffer. Continue to incubate for at least 4 h or longer at room temperature (RT).</li>
		<li>Remove the secondary antibody, wash tissue sections with PBST solution three times, for 10 min each, then wash with clean PBS for another 5 min.</li>
		<li>Wipe off the residual PBS surrounding tissue sections, mount coverslips on sections with mounting medium. Perform fluorescent microscopy to obtain images (see the Table of Materials).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Swamynathan, S. 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. 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=Noggin+overexpression+inhibits+eyelid+opening+by+altering+epidermal+apoptosis+and+differentiation.">Noggin overexpression inhibits eyelid opening by altering epidermal apoptosis and differentiation.</a> The Embo Journal. 22 (12), 2992-3003 (2003).</li><li>Setala, N. L., Metso, J., Jauhiainen, M., Sajantila, A., Holopainen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dry+eye+symptoms+are+increased+in+mice+deficient+in+phospholipid+transfer+protein+(PLTP).">Dry eye symptoms are increased in mice deficient in phospholipid transfer protein (PLTP).</a> The American Journal of Pathology. 178 (5), 2058-2065 (2011).</li><li>Zhang, Y., 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=Mastermind-like+transcriptional+co-activator-mediated+Notch+signaling+is+indispensable+for+maintaining+conjunctival+epithelial+identity.">Mastermind-like transcriptional co-activator-mediated Notch signaling is indispensable for maintaining conjunctival epithelial identity.</a> Development. 140 (3), 594-605 (2013).</li><li>McCauley, 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=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>

The authors have nothing to disclose.

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.
Additional caution should be exercised when handling the lens. The broken pieces of lens are usually scattered through the whole section. This will considerably affect microscopy examination and image analysis. Attempts to remove lens would damage either the ocular surface (if the operation was conducted from facial side) or the retina (if the operation was from the optic disc side of the retina). An alternative way to reduce the negative impact of the presence of lens tissue without dissection is to change sectioning directions. For instance, to save the ocular surface morphology, sectioning of the paraffin block should be performed from anterior of the eyeball to the posterior, otherwise, from the opposite direction.
A useful note to make for the PFA-fixed paraffin section is that antigen retrieval is usually necessary for immunostaining such as using K12 and K14 antibodies. However, there are many exceptions that antigen retrieval would generate non-specific signals increasing the background staining. Thus, one should be cautious when using antigen retrieval technology, and should test antibodies in pilot experiments if possible, beforehand.
In general, there are two methods for isolation of the eyeball from the skull/orbital bones: (i) directly dissect the eyeball from facial side; and (ii) remove the inside skull/orbital bones to free the eyeball. The challenge for the first method is that eyeball sinks deep into the orbital socket with a narrow space in between for dissection. Furthermore, the movable eyeball would stretch the conjunctiva epithelium at any given point when the dissection tools touch it, creating unintentional damages. By contrast, the second method begins with dissection of skull/orbital bones, which are farther from the eyeball and associated ocular surface tissue, thus reducing the risk of damaging the conjunctiva. Moreover, there are no spatial constrictions for performing dissections. Even though the first method can work if great cautions were taken, the second one is definitely easier and better.
In summary, we have described a method for isolation of mouse eyeball with intact eyelid and associated ocular surface from the postnatal mice. The described protocol is suitable for isolation of the eyeball with intact eyelid and associated ocular tissues in postnatal mice. The protocol is especially useful when integrity of the OS or entire ocular tissues is required. We have used this method to prepare tissue samples for examination of keratin markers in OS under normal and pathological conditions. We conclude that the ocular tissues from such preparations are particularly helpful for looking at differential regulations of a protein in different parts of the OS on a single section.

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><tbody><tr><td>1&amp;times; 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&amp;times; PBS and filter with 0.45&amp;mu;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 premaxillary) and posterior (e.g., parietal) as well as ethmoid bones generated tissue block with mainly maxillary and frontal bones atop the eyeball (Figure 1F). Sequential removal of these bones according to designated areas in Figure 1F exposed underlying lacrimal and jugal bones as well as Harderin gland (Figure 1G). The Harderin gland attached to the eyeball served as a landmark (Figure 1G), and should be kept in place at all times. Isolation of the eyeball was completed by removal of all bones and surrounding fats and muscle tissues (Figure 1H).
After paraffin embedding, the eyeball was sectioned and H&#38;E-stained. Representative images are shown in Figure 2. The relative intact positions of eyelids, corneal and conjunctival surface were visualized in one section (Figure 2A), parts of which were magnified in Figure 2B,C. The lens morphology is hard to maintain (Figure 2A) because of its rigidity and fragility. We applied this dissection method to other postnatal ages and showed an example of H&#38;E histology of P10 eyeball section (Figure 3). In general, the younger the mouse is, the better the eyeball morphology is preserved. Immunostained paraffin sections from serial postnatal ages are shown in Figure 4, with keratin 12 (K12) (Figure 4A-F) and keratin 14 (K14) (Figure 4G-L) staining the cornea and the whole OS epithelium, respectively.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60086/60086fig1.jpg" /> 
Figure 1: Illustration of the eyeball dissection in 4-week old adult mouse. (A-D) The schematic diagram of the skull composition viewed from top (A), bottom (B), lateral (C) and middle plane (D), respectively. Dashed lines in (A) and (B) indicate bisected planes. (E) Bisected-half skull at 4-week old age that exactly matches (D). (F) Medial-plane view of dissected tissue between the two transverse planes in (D) and (E). The eyeball (dashed circle) was mainly embedded underneath the frontal (Fron) and maxillary (Max). Colored dashed lines geographically divide the bisected plane into 4 areas, in which bones were roughly removed as per the order or numbers. Note that the ethmoid (Eth) bone has been removed. (G) After removal of frontal bone and partial maxillary, Harderin gland (Har) and jugal (Jug) and lacrimal (La) bones were exposed. Circle dashed lines indicate the eyeball position. (H) Isolated eyeball viewed from inside after dissection completed. Arrow points to optic nerve (On). E = Eye, Na = Nasal, Pre = Premaxilla, Max = Maxillary, La = Lacrimal, Fron = Frontal, Ali = Alisphenoid, Jug = Jugal, Pal = Palatine, Ptery = Pterygoid, Squ = Squamosal, Par = Parietal, Eth = Ethmoid. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60086/60086fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60086/60086fig2.jpg" /> 
Figure 2: H&#38;E histology of the ocular surface tissue at postnatal 4-week old. (A) Eyelid, conjunctiva and cornea are visualized in one tissue section. Boxed areas of &#34;b&#34; and &#34;c&#34; were magnified in (B) and (C), respectively. (B) The cornea epithelium, stroma and endothelium. (C) The conjunctiva with goblet cells. ce = cornea epithelium, st = stroma, ed = endothelium, gc = goblet cell. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60086/60086fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60086/60086fig3.jpg" /> 
Figure 3: H&#38;E histology of the ocular surface tissue at postnatal day 10. (A) Intact eyeball at P10. Boxed areas of &#34;b&#34; and &#34;c&#34; were magnified in (B) and (C), respectively. (B) The cornea. (C) The conjunctiva. Ce = cornea epithelium, st = stroma, ed = endothelium, cj = conjunctiva. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60086/60086fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60086/60086fig4.jpg" /> 
Figure 4: Immunostaining of the ocular surface from P5 to P15. Square-boxed area in each panel was magnified in their right. (A-F) Keratin 12 was specifically expressed in the cornea epithelium (arrows). (G-L) The keratin 14 expressed in the ocular surface. ac = anterior chamber, pc = palpebral conjunctiva. This figure was modified from a previous publication Guo et al.14. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/60086/60086fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Universidad San Sebastian

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JoVE Journal

Biology

8.4K Views.  Sun Yat-sen University. This protocol describes a method for the isolation of the mouse eyeball with eyelid, ocular surface, anterior and posterior segments in relatively intact position.

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

Mouse Eye Enucleation for Remote High-throughput Phenotyping

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular degeneration, photoreceptor degeneration, cataract, glaucoma, retinoblastoma, and diabetic retinopathy have been recapitulated in transgenic mice.1-5 Most transgenic and knockout mice have been generated by laboratories to study non-ophthalmic diseases, but genetic conservation between organ systems suggests that many of the same genes may also play a role in ocular development and disease. Hence, these mice represent an important resource for discovering new genotype-phenotype correlations in the eye. Because these mice are scattered across the globe, it is difficult to acquire, maintain, and phenotype them in an efficient, cost-effective manner. Thus, most high-throughput ophthalmic phenotyping screens are restricted to a few locations that require on-site, ophthalmic expertise to examine eyes in live mice. 6-9 An alternative approach developed by our laboratory is a method for remote tissue-acquisition that can be used in large or small-scale surveys of transgenic mouse eyes. Standardized procedures for video-based surgical skill transfer, tissue fixation, and shipping allow any lab to collect whole eyes from mutant animals and send them for molecular and morphological phenotyping. In this video article, we present techniques to enucleate and transfer both unfixed and perfusion fixed mouse eyes for remote phenotyping analyses.

The dissection technique illustrates enucleation of the mouse eye for tissue fixation to perform phenotyping in high-throughput screens.

The mouse eye is an important genetic model for the translational study of human ophthalmic disease. Blinding diseases in humans, such as macular ...

Medicine

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Developmental Biology

Intravitreal Injections in the Ovine Eye

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or topical administration. IVT drug delivery involves an injection into the vitreous humor of the eye, a gelatinous substance that fills the posterior chamber of the eye and maintains the shape of the eye globe. Although the IVT route is less specifically targeted than subretinal delivery, it is much less invasive and is widely used in clinical settings for a range of ocular diseases. 
We previously demonstrated the efficacy of intravitreal delivery of an adeno-associated virus (AAV)-mediated gene therapy product (AAV9.CLN5) in sheep with a naturally occurring CLN5 form of neuronal ceroid lipofuscinosis (NCL). Affected sheep received IVT gene therapy in one eye, with the other untreated eye serving as an internal control. Retinal structure and function were maintained in the treated eye up to 15 months after treatment, while the untreated eye displayed progressively declining function and severe atrophy during postmortem examination. Based on the sheep studies, the CLN5 gene therapy product was cleared as a candidate investigational new drug (IND) by the United States Food and Drug Administration in September 2021. This paper details the surgical protocol for IVT delivery of a therapeutic viral vector to the ovine eye.

Intravitreal injections were performed in the sheep eye with the aim of delivering viral-mediated gene therapy to the retina.

There are several methods for the delivery of therapeutic agents to the retina, including intravitreal (IVT), subretinal, suprachoroidal, periocular, or ...

Characterization and Morphological Dynamics of Cultured Primary Murine RPE

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important retinal diseases, such as age-related macular degeneration, retinitis pigmentosa, and diabetic retinopathy. We present a streamlined approach for the isolation of RPE from murine adult eyes. In contrast to previously reported methods, this approach enables the isolation and culture of highly pure RPE from adult mice. This simple and fast method does not require extensive technical skill and is achievable with basic scientific tools and reagents. Primary RPE are isolated from C57BL/6 background mice aged 3- to 14-weeks by enucleation of the eye followed by the removal of the anterior segment. Enzymatic trypsinization and centrifugation are used to dissociate and isolate the RPE from the eyecup. In conclusion, this approach offers a quick and effective protocol for the utilization of RPE in the study of retinal function and disease.

Author Spotlight: Efficient Isolation of Primary Retinal Pigment Epithelial Cells from Adult Mice for Retinopathy Research

Retinal pigment epithelium (RPE) acts as a crucial barrier between the choroid and retina, promoting the health and function of retinal cell types, such as photoreceptors. Herein, we describe a simple and effective protocol for isolating and culturing adult murine RPE.

Retinal pigment epithelial cells (RPE) are critical for the proper function of the retina. RPE dysfunction is involved in the pathogenesis of important ...

Neuroscience

Microdissection of the Rodent Eye

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, to obtain the anterior and posterior eyecups. With this technique, the sub-parts of the eye, including the corneal tissue, neural tissue, retinal pigment epithelial (RPE) tissue, and lens, can be obtained for wholemounts, cryo-sectioning, and/or single-cell suspensions of a specific ocular tissue. The presence of the third eyelid presents unique and significant advantages, as it benefits the maintenance of the orientation of the eye, which is important for understanding eye physiology following any localized intervention or in studies involving ocular analysis relating to the eye&#39;s spatial topography.
In this method, we enucleated the eyeball at the socket along with the third eyelid by carefully and slowly cutting through the extraocular muscles and severing the optic nerve. The eyeball was pierced through the corneal limbus using a microblade.&#160;The incision was used as the point of entry, allowing for cutting along the corneal-scleral junction by inserting micro-scissors through the incision point. Small and continuous cuts along the circumference were made until the cups separated. These could be further dissected by gently peeling the translucent layer of the neural retina using Colibri suturing forceps to obtain the neural retina and RPE layers. Further, three/four equidistant cuts were made from the periphery perpendicularly to the optic center until the optic nerve was reached. This opened the hemispherical cups into a floret shape so that they fell flat and could be easily mounted. This technique has been used in our lab for corneal wholemounts and retinal sections. The presence of the third eyelid delineates the nasal-temporal orientation, which allows for the study of various cell therapy interventions post-transplantation and, thus, the targeted physiological validation vital for visualization and accurate representation in such studies.

This paper presents a protocol for ocular micro-dissection in rodents. The process involves the enucleation of the eyeball along with the nictitating membrane (i.e., the third eyelid). This is then followed by the separation of the posterior and anterior eye cups.

The ocular micro-dissection of the rodent eye involves the segmentation of the enucleated eyeball with the attached nictitating membrane, or third eyelid, ...

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular pericytes are central to ophthalmological research. It is vital to choose an appropriate method of visualizing the microvascular pericytes. We describe retinal microvascular pericyte immunohistochemical staining in cryo-sections, whole-mounts, and hypotonic isolated vasculature using antibodies for platelet-derived growth factor receptor &#946; (PDGFR&#946;) and nerve/glial antigen 2 (NG2). This allows us to highlight advantages and shortcomings of each of the three tissue preparations for the visualization of the retinal microvascular pericytes. Cryo-sections provide transsectional visualization of all retinal layers but contain only a few occasional transverse cuts of the microvasculature. Whole-mount provides an overview of the entire retinal vasculature, but visualization of the microvasculature can be troublesome. Hypotonic isolation provides a method to visualize the entire retinal vasculature by the removal of neuronal cells, but this makes the tissue very fragile.

We demonstrate three different tissue preparation techniques for immunohistochemical visualization of rat retinal microvascular pericytes, i.e., cryo-sections, whole-mounts, and hypotonic isolation of the vascular network.

Retinal pericytes play an important role in many diseases of the eye. Immunohistochemical staining techniques of retinal vessels and microvascular ...

Cryosectioning and Immunostaining of Mouse Retina

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. These methods enable detailed examinations of tissue morphologies and the localization of specific proteins within the tissue, which provide valuable insights into disease processes and mechanisms. Mice are the most widely used model for this purpose. However, because mouse eyeballs are small and mouse retinas are extremely delicate tissues, obtaining high-quality retinal sections and immunostaining images from mouse eyeballs is typically challenging. This study describes an improved protocol for cryosectioning mouse retinas and performing immunohistochemistry. An essential point of this protocol involves coating the eyeball with a layer of super glue, which prevents deformation of the eyeballs during the processes of cornea removal, lens extraction, and embedding. This step ensures the integrity of retinal morphologies is well preserved. This protocol highlights critical technical considerations and optimization strategies for consistently producing high-quality retinal sections and achieving excellent immunostaining results.

A protocol for preparing mouse retinal cryosections and performing immunostaining on photoreceptors is described. This article enables researchers to consistently produce mouse retinal frozen sections with well-preserved morphology and high-quality immunostaining results.

Tissue sectioning and immunohistochemistry are essential techniques in histological and pathological studies of retinal diseases using animal models. ...

Assessing Retinal Thickness and Ganglion Cell Layer Counts in Rodent Eyeballs

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many more. Preclinical experiments enable researchers to examine the efficacy of novel drugs, develop new methods of treatment, or seek new pathomechanisms involved in the disease's onset or progression. A histological examination provides a lot of information necessary to assess the effects of the conducted experiments and can reveal degeneration, tissue remodeling, infiltration, and many other pathologies. In clinical research, there is rarely any chance of obtaining eye tissue suitable for a histological examination, which is why researchers should take advantage of the opportunity offered by the examination of eyeballs from rodents. This manuscript presents a protocol for the histological preparation of rodent eyeballs' sections. The procedure is presented for the eyeballs of mice and rats and has the following steps: (i) harvesting the eyeball, (ii) preserving the eyeball for further analysis, (iii) processing the tissue in paraffin, (iv) preparing slides, (v) staining with hematoxylin and eosin, (vi) assessing the tissue under a light microscope. With the proposed method, the retina can be easily visualized and assessed in detail.

This manuscript presents a protocol for the histological preparation of slides from rodent eyeballs. With the proposed method, the inner retina can be easily visualized and assessed under a light microscope. The procedure is presented for the eyeballs of mice and rats.

A rodent eyeball is a powerful tool for researching the pathomechanisms of many ophthalmic diseases, such as glaucoma, hypertensive retinopathy, and many ...

Rabbit Eye Dissection and Cryosectioning for Immunohistochemistry Studies

Obtaining High-Quality Cryosections of Whole Rabbit Eye

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed in the fixative. Then, the cornea and iris are removed and the eye is left overnight for additional fixation at 4 &#176;C. Following fixation, the lens is removed. The eye is then placed in a cryomold and filled with an embedding medium. By removing the lens, the embedding medium has better access to the vitreous and leads to better retinal stability. Importantly, the eye should be incubated in embedding medium overnight to allow complete infiltration throughout the vitreous. Following overnight incubation, the eye is frozen on dry ice and sectioned. Whole retinal sections may be obtained for use in immunohistochemistry. Standard staining protocols may be utilized to study the localization of antigens within the retinal tissue. Adherence to this protocol results in high-quality retinal cryosections that may be used in any experiment utilizing immunohistochemistry.

Author Spotlight: Optimizing Retinal Tissue Processing for Immunohistochemistry in Rabbit Models

This protocol describes a reliable method for obtaining high-quality cryosections of whole rabbit eyes. It details rabbit eye dissection, fixation, embedding, and sectioning procedures, which may be easily adapted for use in any study utilizing immunohistochemistry in larger eyes.

This protocol describes how to obtain high-quality retinal cryosections in larger animals, such as rabbits. After enucleation, the eye is briefly immersed ...

A Cost-Effective Method for Isolating Primary RPE Cells from Porcine Eyes

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly occurs in diseases marked by progressive vision loss, such as age-related macular degeneration (AMD). Research on AMD often relies on human donor eyes or induced pluripotent stem cells (iPSCs) to represent the RPE. However, these RPE sources require extended differentiation periods and substantial expertise for culturing. Additionally, some research institutions, particularly those in rural areas, lack easy access to donor eyes. While a commercially available immortalized RPE cell line (ARPE-19) exists, it lacks essential in vivo RPE features and is not widely accepted in many ophthalmology research publications. There is a pressing need to obtain representative primary RPE cells from a more readily available and cost-effective source. This protocol elucidates the isolation and subculture of primary RPE cells obtained post-mortem from porcine eyes, which can be sourced locally from commercial or academic suppliers. This protocol necessitates common materials typically found in tissue culture labs. The result is a primary, differentiated, and cost-effective alternative to iPSCs, human donor eyes, and ARPE-19 cells.

Author Spotlight: Modeling Retinal Pathologies with Enhanced RPE Cell-Based Disease Models

This protocol outlines the procedure for obtaining and culturing primary retinal pigment epithelial (RPE) cells from locally sourced porcine eyes. These cells serve as a high-quality alternative to stem cells and are suitable for in vitro retinal research.

The retinal pigment epithelium (RPE) is a crucial monolayer in the outer retina responsible for supporting photoreceptors. RPE degeneration commonly ...

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

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Chapters in this video

Mouse Eye Enucleation for Remote High-throughput Phenotyping

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

Intravitreal Injections in the Ovine Eye

A Simplified Method for Isolation and Culture of Retinal Pigment Epithelial Cells from Adult Mice

Microdissection of the Rodent Eye

Retinal Cryo-sections, Whole-Mounts, and Hypotonic Isolated Vasculature Preparations for Immunohistochemical Visualization of Microvascular Pericytes

Cryosectioning and Immunostaining of Mouse Retina

Preparation and Analysis of Histological Slides of Rat and Mouse Eyeballs to Evaluate the Retina

Obtaining High-Quality Cryosections of Whole Rabbit Eye

Isolation of Primary Porcine Retinal Pigment Epithelial Cells for In Vitro Modeling