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....

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. ...

isolation-intact-eyeball-to-obtain-integral-ocular-surface-tissue-for

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8.3K 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

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Production of Tissue Microarrays, Immunohistochemistry Staining and Digitalization Within the Human Protein Atlas

The tissue microarray (TMA) technology provides the means for high-throughput analysis of multiple tissues and cells. The technique is used within the Human Protein Atlas project for global analysis of protein expression patterns in normal human tissues, cancer and cell lines. Here we present the assembly of 1 mm cores, retrieved from microscopically selected representative tissues, into a single recipient TMA block. The number and size of cores in a TMA block can be varied from approximately forty 2 mm cores to hundreds of 0.6 mm cores. The advantage of using TMA technology is that large amount of data can rapidly be obtained using a single immunostaining protocol to avoid experimental variability. Importantly, only limited amount of scarce tissue is needed, which allows for the analysis of large patient cohorts 1 2. Approximately 250 consecutive sections (4 &mu;m thick) can be cut from a TMA block and used for immunohistochemical staining to determine specific protein expression patterns for 250 different antibodies. In the Human Protein Atlas project, antibodies are generated towards all human proteins and used to acquire corresponding protein profiles in both normal human tissues from 144 individuals and cancer tissues from 216 different patients, representing the 20 most common forms of human cancer. Immunohistochemically stained TMA sections on glass slides are scanned to create high-resolution images from which pathologists can interpret and annotate the outcome of immunohistochemistry. Images together with corresponding pathology-based annotation data are made publically available for the research community through the Human Protein Atlas portal (<a href="http://www.proteinatlas.org" target="_blank">www.proteinatlas.org</a>) (Figure 1) 3 4. The Human Protein Atlas provides a map showing the distribution and relative abundance of proteins in the human body. The current version contains over 11 million images with protein expression data for 12.238 unique proteins, corresponding to more than 61% of all proteins encoded by the human genome.

Tissue microarrays allows for an efficient method to gain concurrent information from a multitude of tissues. Representative parts of tissues are assembled into a single paraffin block. Sections from the block are used for immunohistochemistry and analysis of protein expression patterns. Digital scanning generates corresponding images for distribution of data.

The  tissue microarray (TMA) technology provides the means for high-throughput  analysis of multiple tissues and cells. The technique is used within the ...

Isolation and Differentiation of Stromal Vascular Cells to Beige/Brite Cells

Brown adipocytes have the ability to uncouple the respiratory chain in mitochondria and dissipate chemical energy as heat. Development of UCP1-positive brown adipocytes in white adipose tissues (so called beige or brite cells) is highly induced by a variety of environmental cues such as chronic cold exposure or by PPAR&gamma; agonists, therefore, this cell type has potential as a therapeutic target for obesity treatment. Although most immortalized adipocyte lines cannot recapitulate the process of "browning" of white fat in culture, primary adipocytes isolated from stromal vascular fraction in subcutaneous white adipose tissue (WAT) provide a reliable cellular system to study the molecular control of beige/brite cell development. Here we describe a protocol for effective isolation of primary preadipocytes and for inducing differentiation to beige/brite cells in culture. The browning effect can be assessed by the expression of brown fat-selective markers such as UCP1.

Primary white preadipocytes isolated from white adipose tissues in mice can be differentiated into beige/brite cells. Presented here is a reliable cellular model system to study the molecular regulation of "browning" of white fat.

Brown adipocytes have the ability to uncouple the  respiratory chain in mitochondria and dissipate chemical energy as heat.  Development of UCP1-positive ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Histological-Based Stainings Using Free-Floating Tissue Sections

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative approaches are widely used to handle the tissue sections during the staining procedure, one approach consists of mounting the sections directly on glass slides, while a second approach, the free-floating, allows for fixed sections to be maintained and stained while suspended in solution. Although slide-mounted and free-floating approaches may yield similar results, the free-floating technique allows for better antibody penetration and thus should be the method of choice when thicker sections are to be used for 3D reconstruction of the tissues, for example when the focus of the experiment is to gain information on dendritic and axonal projections in brain regions. In addition, since the sections are kept in solution, a single aliquot can easily accommodate 30 to 40 sections, handling of which is less laborious, particularly in large-scale biomedical studies. Here, we illustrate how to apply the free-floating method to fluorescent immunohistochemistry staining, with a major focus on brain sections. We will also discuss how the free-floating technique can easily be modified to fit the individual needs of researchers and adapted to other tissues as well as other histochemical-based stainings, such as hematoxylin and eosin and cresyl violet, as long as tissue samples are properly fixed, typically with paraformaldehyde or formalin.

The free-floating technique allows researchers to perform histological-based stainings including immunohistochemistry on fixed tissue sections to visualize biological structures, cell type, and protein expression and localization. This is an efficient and reliable histochemical technique that can be useful for investigating a multitude of tissues, such as brain, heart, and liver.

Immunohistochemistry is a widely used technique to visualize specific tissue structures as well as protein expression and localization. Two alternative ...

Isolation and Characterization of Intact Phycobilisome in Cyanobacteria

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. Studying the structure and composition of phycobilisome is of great interest to scientists because it reveals the evolution and divergence of photosynthesis in cyanobacteria. This protocol provides a detailed and optimized method to break cyanobacterial cells at low cost by a bead-beater efficiently. The intact phycobilisome can then be isolated from the cell extract by sucrose gradient ultracentrifugation. This method has demonstrated being suitable for both model and non-model cyanobacteria with different cell types. A step-by-step procedure is also provided to confirm the integrity and property of phycobiliproteins by 77K fluorescence spectroscopy and SDS-PAGE stained by zinc sulfate and Coomassie Blue. The isolated phycobilisome can also be subjected to further structural and compositional analyses. Overall, this protocol provides a helpful starting guide that allows researchers unfamiliar with cyanobacteria to quickly isolate and characterize intact phycobilisome.

The current protocol details the isolation of phycobilisomes from cyanobacteria by centrifugation through a discontinuous sucrose density gradient. The fractions of intact phycobilisomes are confirmed by 77K fluorescent emission spectrum and SDS-PAGE analysis. The resulting phycobilisome fractions are suitable for negative staining of TEM and mass spectrometry analysis.

In cyanobacteria, phycobilisome is a vital antenna protein complex that harvests light and transfers energy to photosystem I and II for photochemistry. ...

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

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 ...