The authors would like to thank E. de la Rosa and A.I. Valenciano for initial help with the establishment of the organotypic cultures and U. Laub and U. Gerster for technical assistance.

All equipment and reagents have to be purchased sterile or needs to be heat or steam sterilized or sterilized with 70% ETOH.
The authors state that experiments on animals were performed in accordance with the European Communities Council Directive (86/609/EEC), following the Guidelines of the NIH regarding the care and use of animals for experimental procedures and the regulations set forth by the Institutional Animal Care and Use Committee (IACUC) at the University of Duisburg-Essen (Germany).
Part 1: Enucleation of murine eyes of different developmental stages
Enucleation of embryonic eyes
<ol>
 <li>Time pregnant matings are set up and the morning of the day on which a vaginal plug is detected in females mating is designated gestation day 0.</li>
 <li>The pregnant female is sacrificed by cervical dislocation when the development of the embryos has reached the desired stage (here: embryonic day (E) 15) and fixed on a wax board6.</li>
 <li>The abdominal wall is moistened with 70% ETOH, cut along the midline and the skin flaps are fixed laterally by pins6.</li>
 <li>The uterusses are removed from the abdomen, detached and transferred to a beaker with cold PBS6.</li>
 <li>The embryos are separated, transferred to a Petri dish and uterus wall and embryonic membranes are removed carefully by the use of forceps6. </li>
 <li>Embryos are killed by decapitation.</li>
 <li>Eyes are enucleated be the use of fine, curved forceps, "peeling" the eyes from the eye socket.</li>
</ol>
Enucleation of postnatal and adult eyes
<ol start="1" type="1">
 <li>Young pups are killed by decapitation, older pups and adults by cervical dislocation.</li>
 <li>Up to postnatal stage P15, the time point when mice open their eyes, eye slits have to be mechanically opened by the use of forceps and enlarged by two transverse cuts of the eye lids with spring scissors.</li>
 <li>Eyes are enucleated by the help of curved forceps, applying pressure to the orbit.</li>
</ol>
Note: As at postnatal day 2, the orbital bones are still cartilaginous, it is important not to apply too much pressure while trying to remove the eyes. 
 By contrast, in the adult mice, the orbital bones are firm. Thus, in order to enucleate the eyes it is sufficient to apply pressure to the orbit without enlarging the eye slits in advance.
Part 2: Dissection of embryonic, postnatal and adult murine retinas
Dissection of retinas
<ol>
 <li>The eyes are placed in a small Petri dish with sterile PBS and the surrounding eye layers are removed under a dissection microscope.</li>
 <li>To remove the outer eye layers, in postnatal stages and adult eyes the optic nerve has to be cut off by the help of spring scissors or pinched off by forceps as close to the basis as possible.</li>
 <li>Turn the eye, so that the back side with the hole where the optic nerve originally resided is facing you. Enter the subretinal space between retina and pigment epithelium with the tips of two very fine forceps from the site where the optic nerve penetrated the eye layers. 
Note: Usually, the pigment epithelium can be easily identified by its dark colour. In some mouse mutant strains - especially in albino animals - this pigment layer may however, not be coloured and thus may not be readily to detect.</li>
 <li>Remove the pigment epithelium with the attached choroid membrane and sclera by carefully tearing to either side with both forceps. </li>
 <li>Peel off the layers up to the level of the cornea, then turn the eye cup to the lens side and remove the cornea together with pigment epithelium, choroid membrane and sclera, while holding up the remaining retinal cup by the other forceps.</li>
 <li>Grasp the vitreous together with the small lens and while tearing with forceps, keep the retinal wholemount in place with the second forceps. 
Note: When dissecting embryonic eyes, make sure to completely remove the triangular, tent-like capillary plexus underneath the vitreous body together with the vitreous. 
In the adult eye, the vitreous needs to be grasped at the sides and care has to be taken not to pierce the vitreous with the tips of the forceps as its content is viscous and sticks to the forceps, hindering its removal.</li>
 <li>For organotypic wholemount culture the retinal cups are collected in a 96-well plate containing 200 &mu;l Dulbecco's modified eagle's medium (see below). </li>
</ol>
Note: Between dissection of individual retinas, keep the 96-well collection plate containing the culture medium in the incubator as the pH of the culture medium is triggered by CO2 via the carbonate system.
Part 3: Murine organotypic retinal wholemount culture
<ol>
 <li>Prepare 500 ml culture by weighting 7.8g Dulbecco's modified eagle's medium / nutrient mixture F-12 HAM (DMEM) and 0.6g NAHCO3 and dissolving both in MiIliQ water. Adjust the pH to 7.15. Add 50mg apo-transferin, 50&mu;l putrescin (stock: 60mg/ml), 50&mu;l sodium selenite (stock: 52&mu;g/ml), progesterone (stock: 60&mu;g/ml), and 2.5 ml gentamicine (200 mM) under the hood. Mix and filter through a bottle top filter. Immediately before usage add 10&mu;l glutamine (200 mM) per ml culture medium. 
Note: This serum- and insulin-free medium can be stored at 4&deg;C for up to 2 weeks and used for apoptosis induction experiments as no insulin counteracts the effects. If incubation of wholemounts for longer than 24h is desired and cell death rates will not be evaluated, insulin serum (e.g. fetal calf serum; FCS) or supplements should be added to improve survival rates.</li>
 <li>Before starting the culture, the retinal wholemounts are pre-incubated for 15 min at 37&deg;C with 200 &mu;l warm, pH-balanced DMEM containing 0.5mg/ml hyaluronidase to pre-digest the hyaluronidase containing inner and outer limiting membrane of M ller glia cells, facilitating penetration of exogenously applied substances. </li>
 <li>Retinas are transferred to a 24-well plate with as few hyaluronidase as possible and cultured as organotypic wholemounts in 2ml chemically defined Dulbecco's modified eagle medium. 
Note: For transferring the retinas, use a 1ml pipette and cut the pipette tip a few millimetres to widen the opening. For embryonic eyes, a 200 &mu;l pipette tip is sufficient. The cutting edges should be smoothened by insertion and twisting of a second pipette tip. 
 For 24-48 hrs short-term cultures, all steps can be performed at the bench, but if contamination of the cultures turns out to be a problem, one should work under the hood.</li>
 <li>Cultures are maintained for 24 - 48 hrs at 37&deg;C in a 5% CO2 atmosphere and e.g. subjected to pharmacological treatment with recombinant factors.</li>
</ol>
Part 4: Dissociation of cultured retinal wholemounts
<ol>
 <li>After the desired culture time, retinas are collected in 2ml Eppendorf tubes with round bottom containing 850 &mu;l PBS and 50 &mu;l bovine serum albumine (BSA; 30 mg/ml).</li>
 <li>Place Eppendorf tubes with retinas into a heating block, heated up at 37&deg;C.</li>
 <li>Add 25 &mu;l collagenase (200 U/ml) and 25 &mu;l hyaluronidase (20mg/ml) to each Eppendorf tube and start dissociating the retinas into single cell suspension by 3 passes through a siliconized Pasteur pipette. </li>
 <li>Add 10 &mu;l trypsin (1mg/ml), wait for 3-5 min, and then slowly pipette 3-5 times up and down with siliconized Pasteur pipette to mechanically dissociate the tissue.</li>
 <li>Add 10 &mu;l DNase I (5mg/ml), again wait for 3-5 min, then slowly pipette 3-5 times up and down with siliconized Pasteur pipette. 
Note: The incubation time for enzymatic dissociation varies and depends on the size of the eyes and the developmental stage, respectively. Check the stage of enzymatic digestion of the tissue by gently pipetting up and down.</li>
 <li>If the cell suspension is not homogenous by now but still contains major cell aggregates, add additional 10 &mu;l trypsin and 10 &mu;l DNase I.</li>
 <li>When the cell suspension is homogeneous, digestion of the tissue is stopped by addition of 10 &mu;l EDTA (0.5 M), Eppendorf tubes are removed from the heater and the cell suspensions are fixed for 1h by addition of 1ml fresh, ice cold 8% paraformaldehyde (PFA) at room temperature on a rotation shaker.</li>
</ol>
Part 5: Washing of dissociated cell suspensions
<ol>
 <li>The cell suspension is centrifuged for 5 min at 4&deg;C and at 0.2 rcf in a cooling centrifuge.</li>
 <li>The supernatant is discarded and the pellet is re-suspended in 1ml PBS containing 3mg/ml BSA. </li>
 <li>After repeating these washing steps twice, the pellet is finally re-suspended in 500 &mu;l PBS containing 3mg/ml BSA, 5mM EDTA and 0.1% sodium azide.</li>
</ol>
Note: The addition of sodium acid allow storage of the cell suspension for several days at 4&deg;C. However, if an immunocytochemical staining will follow, do not add sodium acid to the resuspension buffer, as this results in loss of staining quality.
Part 6: Cytospin of cell suspensions for quantitative apoptosis and proliferation analysis
<ol>
 <li>A frosted-end microscope slide, a cytospin filter with one or two holes and a cytospin funnel are inserted into a cytospin slide clip. The slide clip is closed and positioned in the cytospin rotor. </li>
 <li>The dissociated cell suspension is homogenized by gently pipetting up and down. Note: Depending on the developmental stage of the retina, the cell suspension from the dissociation procedure might need to be diluted with PBS to obtain a countable number of cells.
</li>
 <li>An aliquot (100 &mu;l) of the cell suspension is applied to a cytospin funnel. 
Note: When pipetting the cell suspension to the funnel, the tip of the pipette should reach all the way down to the bottom of the funnel. It is important not to push through the second pressure point of the pipette as this creates air bubbles, which will be visible in the cell spot after the cytospin and hampers cell counts.</li>
 <li>The cell suspension is spotted on a slide at 700 rpm for 7 min. </li>
 <li>For determination of the effect of exogenously applied factors on apoptosis levels cells can be stained with 4',6-Diamidino-2-phenylindole (DAPI; 2&#956;g/ml), mounted with fluorescent mounting medium. Changes in cell apoptosis can be determined by counting at least 1000 cells (comprising at least 10 pycnotic nuclei) in the cytospin cell spots and the cell death rate is calculated as percentage of total cells counts3,4.
 Note: Alternatively, the distribution of apoptotic nuclei can be evaluated in flatmounts3 or cryostat sections4 of cultured retinal wholemounts by TdT-mediated dUTP nick end labeling (TUNEL).</li>
 <li>For detection of cell proliferation, BrDU (5 &mu;M) can be added 6h before end of the culture and BrdU incorporation visualized in cytospins of cell homogenates by immunocytochemical staining using an anti-BrDU antibody (e.g. Developmental Studies Hybridoma Bank, Iowa, USA). </li>
 <li>The effect of treatment on different retinal cell types can be visualized in cytospins by neuron specific antibodies like Brn3a (ganglion cell marker) or opsin (photoreceptor marker) and counterstaining with DAPI. </li>
</ol>
Part 7: Representative Results
<img src="/files/ftp_upload/1634/1634fig1tmb.jpg" border="0" alt="Figure 1" /> Figure 1: Steps in preparation of murine organotypic retinal wholemounts A Head of mouse with both eyes. B Murine eye with lens side up, all layers still in place. C Murine eye from the backside with the optic nerve still attached. D Murine eye with sclera and pigment epithelium partially removed. E Murine retina with cornea, sclera and pigment epithelium completely removed, but lens and vitreous still in place. F Murine retinal wholemount cup with lens and vitreous removed. Please <a href="https://www.jove.com/files/ftp_upload/1634/1634fig1.jpg">click here</a> to see a larger version of figure 1.
<img src="/files/ftp_upload/1634/1634fig2tmb.jpg" border="0" alt="Figure 2" /> Figure 2: Analysis of organotypic retinal wholemount cultures by cytospin and sections For analysis of apoptosis, cytospins of dissociated cell suspensions are stained by DAPI and pycnotic nuclei can be distinguished by nuclear fragmentation or chromatin condensation (arrowheads in A). Alternatively, wholemount sections (C-E; murine retina postnatal day (P) 2) or retinal flatmount (F) can be subjected to a TUNEL assay and counterstained with DAPI (E). The effect of treatment on different retinal cell types can be visualized in cytospins by neuron specific antibodies like the ganglion cell marker Brn3a (arrows in B). GCL, ganglion cell layer; INL, prospective inner nuclear layer. Please <a href="https://www.jove.com/files/ftp_upload/1634/1634fig2.jpg">click here</a> to see a larger version of figure 2.

<ol>
	<li>Rieke, M., Gottwald, E., Weibezahn, K. -. F., Layer, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+reconstruction+in+3D-spheroids+from+rodent+retina+in+a+motion-free,+bioreactor-based+microstructure.">Tissue reconstruction in 3D-spheroids from rodent retina in a motion-free, bioreactor-based microstructure.</a> Lab. Chip. 8, 2006-2213 (2008).</li><li>Donovan, S. L., Dyer, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+square+wave+electroporation+of+retinal+explant+cultures.">Preparation and square wave electroporation of retinal explant cultures.</a> Nature Protocols. 1, 2710-2718 (2006).</li><li>Duenker, N., Valenciano, A. I., Franke, A., Hernandez-Sanchez, C., Dressel, R., Behrendt, M., de Pablo, F., Krieglstein, K., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Balance+of+pro-apoptotic+transforming+growth+factor-beta+and+anti-apoptotic+insulin+effects+in+the+control+of+cell+death+in+the+postnatal+mouse+retina.">Balance of pro-apoptotic transforming growth factor-beta and anti-apoptotic insulin effects in the control of cell death in the postnatal mouse retina.</a> Eur. J. Neurosci. 22, 28-38 (2005).</li><li>Franke, A. G., Gubbe, C., Beier, M., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factors+beta+and+Bone+morphogenetic+proteins:+Cooperative+players+in+chick+and+murine+programmed+retinal+cell+death.">Transforming growth factors beta and Bone morphogenetic proteins: Cooperative players in chick and murine programmed retinal cell death.</a> J. Comp. Neurol. 495, 263-278 (2005).</li><li>de la Rosa, E. J., D&#237;az, B., De Pablo, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoculture+of+the+chick+embryonic+neuroretina.">Organoculture of the chick embryonic neuroretina.</a> Curr. Top. Dev. Biol. 36, 133-144 (1998).</li><li>Dohle, D. S., Pasa, S. D., Gustmann, S., Laub, M., Wissler, J. H., Jennissen, H. P., Duenker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+How+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: How it really works.</a> J Vis Exp. 32, (2009).</li></ol>

The advantage of murine organotypic retinal wholemount cultures2-5 over dissociation, monolayer, retinospheroid or re-aggregated 3D spheroid cultures1 lies in the preservation of neuronal interactions and connections, mimicking the in vivo situation. In comparison to former reports2, our video article provides a detailed demonstration of the peculiarities in enucleation of murine eyes and dissection of retinas of different developmental stages including removal of lens and vitreo...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Mice</td>
<td>Animal</td>
<td>Charles Rivers Laboratories</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>Tool</td>
<td>ZEISS</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>Reagent</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>PBS should be cold (&gt; 4&deg;C) and sterile</td>
</tr>
<tr>
<td>Dulbecco`s modified eagle`s medium / nutrient mixture F-12 Ham</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 8900</td>
<td>DMEM / F-12</td>
</tr>
<tr>
<td>Apo-transferin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T 1147</td>
<td>&nbsp;</td>
<tr>
<td>Putrescin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 5780</td>
<td>&nbsp;</td>
<tr>
<td>Sodium selenite</td>
<td>Reagent</td>
<td>Sigma</td>
<td>S 9133</td>
<td>&nbsp;</td>
<tr>
<td>Progesterone</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P 6149</td>
<td>&nbsp;</td>
<tr>
<td>Gentamicine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamine</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>25030-024</td>
<td>200 mM (100X), liquid</td>
</tr>
<tr>
<td>Bovine serum albumine (BSA)</td>
<td>Reagent</td>
<td>Roth</td>
<td>8076.3</td>
<td>30 mg/ml</td>
</tr>
<tr>
<td>Collagenase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>C 0773</td>
<td>200 U/ml</td>
</tr>
<tr>
<td>Trypsin</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T4799</td>
<td>From porcine pancreas; 1 mg/ml</td>
</tr>
<tr>
<td>Hyaluronidase</td>
<td>Reagent</td>
<td>Sigma</td>
<td>H 3884</td>
<td>200 mg/ml</td>
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<tr>
<td>DNase I</td>
<td>Reagent</td>
<td>Roche</td>
<td>1 284 932</td>
<td>10 mg/ml</td>
</tr>
<tr>
<td>EDTA</td>
<td>Reagent</td>
<td>Sigma</td>
<td>E 6511</td>
<td>&nbsp;</td>
<tr>
<td>Silicone solution</td>
<td>Reagent</td>
<td>Serva</td>
<td>35130</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P6148</td>
<td>8% PFA in 0.1M phosphate buffer (pH 7.4).</td>
</tr>
<tr>
<td>4',6-diamidino-2-phenylindole dihydrochloride</td>
<td>Reagent</td>
<td>Sigma</td>
<td>D 0542</td>
<td>DAPI</td>
</tr>
<tr>
<td>Fluorescent Mounting Medium</td>
<td>Reagent</td>
<td>Dako</td>
<td>S3023</td>
<td>&nbsp;</td>
<tr>
<td>BrDU</td>
<td>Reagent</td>
<td>Sigma</td>
<td>B 9285</td>
<td>&nbsp;</td>
<tr>
<td>96-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3072</td>
<td>&nbsp;</td>
<tr>
<td>24-well plates</td>
<td>Tool</td>
<td>FALCON</td>
<td>3047</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur pipettes</td>
<td>Tool</td>
<td>Brand</td>
<td>747720</td>
<td>&nbsp;</td>
<tr>
<td>Forceps DUMONT #5</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-30</td>
<td>bevelled very fine shanks (0.05 mm x 0.02 mm tip)</td>
</tr>
<tr>
<td>Forceps DUMONT #7</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11271-30</td>
<td>curved shanks (0.07 mm x 0.10 mm tip)</td>
</tr>
<tr>
<td>Spring scissors,straight, 8cm</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>15000-00</td>
<td>fine, small straight blades</td>
</tr>
<tr>
<td>Standard scissors, straight, sharp/blunt </td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14007-14</td>
<td>Use for decapitation or cervical dislocation</td>
</tr>
<tr>
<td>Eppendorf tubes</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>2ml; round bottom for better precipitation of pellet during centrifugation /cytospin</td>
</tr>
<tr>
<td>Cooling centrifuge</td>
<td>Tool</td>
<td>Eppendorf</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Rotation shaker</td>
<td>Tool</td>
<td>CAT</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Cytospin</td>
<td>Tool</td>
<td>Thermo Scientific</td>
<td>&nbsp;</td>
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in vivo-like organotypic murine retinal wholemount culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, retina-specific or conditional knockout mouse models often display early lethality or suffer from severe malformations, preventing an analysis beyond embryonic or early postnatal stages.
Primary cell culture is an alternative to investigate the effects of exogenously applied recombinant factors, overexpression of genes or siRNA-mediated gene knockdown in a controlled environment. Dissociated cell culture has the advantage that the endogenous signals reaching the target cells are reduced, thereby facilitating the identification of exogenously triggered effects after pharmacological manipulation. However, important cell-cell interactions are initially destroyed by enzymatic digestion or mechanical dissociation, even if re-aggregated retinospheroid cultures1 are used.
By contrast, organotypic retinal wholemount cultures provide a system close to the physiological in vivo situation with neuronal interactions and connections still preserved2-5.
In this video article we provide a step by step demonstration of (1) the establishment of in vivo-like organotypic retinal wholemount cultures including dissection peculiarities of embryonic, postnatal and adult murine eyes and (2) a dissociation and cytospin procedure for analysis of neuronal apoptosis and retinal cell proliferation in organotypic wholemounts, e.g. after culture in the presence of exogenously applied recombinant factors.

Watch this Scientific Journal Video about In vivo-like Organotypic Murine Retinal Wholemount Culture at JoVE.com

In vivo-like Organotypic Murine Retinal Wholemount Culture

This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

In vivo-like Organotypic Murine Retinal Wholemount Culture

Targeted ablations of genes and analysis of animal models is the classical strategy for enrolling specific retinal gene function. However, transgenic, ...

in-vivo-like-organotypic-murine-retinal-wholemount-culture

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JoVE Journal

Biology

17.7K Views.  University of Duisburg-Essen. This video article demonstrates the establishment of organotypic retinal wholemount cultures and a cytospin procedure for analysis of exogenously induced effects. Organotypic retinal wholemount cultures mimic the in vivo situation and significantly facilitate the accessibility of murine retinas for experimental manipulations while circumventing the disadvantages of classical murine animal models.

Video: In vivo-like Organotypic Murine Retinal Wholemount Culture

Organotypic Culture of Adult Rabbit Retina

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with imaging techniques, genetic manipulation, and electrophysiological recording. Here we present a simple interphase tissue culture system for adult rabbit retina that requires no specialized equipment and very little maintenance. We demonstrate the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells. Rabbit retinas cultured this way can be kept alive for up to 6 days with very little changes of the overall anatomical structure or the morphology of individual ganglion- and amacrine cells.

This article demonstrates the dissection and incubation of rabbit retina and particle-mediated gene transfer of plasmids encoding GFP or a variety of subcellular markers into retinal ganglion cells.

Organotypic culture systems of functional neural tissues are important tools in neurobiological research. Ideally, such a system should be compatible with ...

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Whole-cell Recordings of Light Evoked Excitatory Synaptic Currents in the Retinal Slice

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we will guide you through the process of performing whole-cell recordings of light evoked currents of individual cells in the retinal slice preparation. We use the aquatic tiger salamander as an animal model. We begin by describing the dissection of the eye and show how slices are mounted for electrophysiological recordings. Once the slice is placed in the recording chamber, we demonstrate how to perform whole-cell voltage clamp recordings. We then project visual stimuli onto the photoreceptors in the slice to elicit light-evoked current responses. During the recording we perfuse the slice with pharmacological agents, whereby an 8-channel perfusion system allows us to quickly switch between different agents.  The retinal slice preparation is widely used for patch clamp recordings in the retina, in particular to study amacrine or bipolar cells, which are not accessible in a whole-mount preparation.

This video shows the process of whole-cell voltage clamp recordings in the retinal slice of the aquatic tiger salamander. We demonstrate the preparation of the slice as well as how to perform patch clamp recordings during visual stimulation of the retina.

We use the whole-cell patch clamp technique to study the synaptic circuitry that underlies visual information processing in the retina. In this video, we ...

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

Measurement of Energy Metabolism in Explanted Retinal Tissue Using Extracellular Flux Analysis

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while maintaining transparency of the visual axis. Perturbations to this delicate balance cause blinding illnesses, such as diabetic retinopathy. Therefore, the understanding of energy metabolism changes in the retina during disease is imperative to the development of rational therapies for various causes of vison loss. The recent advent of commercially-available extracellular flux analyzers has made the study of retinal energy metabolism more accessible. This protocol describes the use of such an analyzer to measure contributions to retinal energy supply through its two principle arms - oxidative phosphorylation and glycolysis - by quantifying changes in oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) as proxies for these pathways. This technique is readily performed in explanted retinal tissue, facilitating assessment of responses to multiple pharmacologic agents in a single experiment. Metabolic signatures in retinas from animals lacking rod photoreceptor signaling are compared to wild-type controls using this method. A major limitation in this technique is the lack of ability to discriminate between light-adapted and dark-adapted energy utilization, an important physiologic consideration in retinal tissue.

This technique describes real time recording of oxygen consumption and extracellular acidification rates in explanted mouse retinal tissues using an extracellular flux analyzer.

High acuity vision is a heavily energy-consuming process, and the retina has developed several unique adaptations to precisely meet such demands while ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...