This work was supported by NIH grant R01-EY020535 (B.T.S), International Retinal Research Foundation and Karl Kirchgessner Foundation (B.T.S).

In all experimental procedures, animals were treated according to the regulations in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and in compliance with protocols approved by Weill Cornell Medical College. Animals were euthanized by carbon dioxide and subsequent cervical dislocation.
1. Dissection of Wholemount Retina
<ol>
	<li>After the mouse is euthanized, enucleate its eyes and place them into oxygenated HEPES-buffered Ringer&#39;s solution (Table 1<...

<ol>
	<li>Csaba, G., Kovacs, P., Pallinger, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EDAC+fixation+increases+the+demonstrability+of+biogenic+amines+in+the+unicellular+Tetrahymena:+a+flow+cytometric+and+confocal+microscopic+comparative+analysis.">EDAC fixation increases the demonstrability of biogenic amines in the unicellular Tetrahymena: a flow cytometric and confocal microscopic comparative analysis.</a> Cell Biol. Int. 30, 345-348 (2006).</li><li>Fletcher, E. L., Hack, I., Brandstatter, J. H., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+localization+of+NMDA+receptor+subunits+in+the+rat+retina.">Synaptic localization of NMDA receptor subunits in the rat retina.</a> J. Comp. Neurol. 420, 98-112 (2000).</li><li>Gastinger, M. J., O'Brien, J. J., Larsen, N. B., Marshak, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histamine+immunoreactive+axons+in+the+macaque+retina.">Histamine immunoreactive axons in the macaque retina.</a> Invest. Ophthalmol. Vis. Sci. 40, 487-495 (1999).</li><li>Haverkamp, S., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+analysis+of+the+mouse+retina.">Immunocytochemical analysis of the mouse retina.</a> J. Comp. Neurol. 424, 1-23 (2000).</li><li>Koulen, P., Fletcher, E. L., Craven, S. E., Bredt, D. S., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunocytochemical+localization+of+the+postsynaptic+density+protein+PSD-95+in+the+mammalian+retina.">Immunocytochemical localization of the postsynaptic density protein PSD-95 in the mammalian retina.</a> J. Neurosci. 18, 10136-10149 (1998).</li><li>Pena, J. T., 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=miRNA+in+situ+hybridization+in+formaldehyde+and+EDC-fixed+tissues.">miRNA in situ hybridization in formaldehyde and EDC-fixed tissues.</a> Nat. Methods. 6, 139-141 (2009).</li><li>Tymianski, M., 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+novel+use+for+a+carbodiimide+compound+for+the+fixation+of+fluorescent+and+non-fluorescent+calcium+indicators+in+situ+following+physiological+experiments.">A novel use for a carbodiimide compound for the fixation of fluorescent and non-fluorescent calcium indicators in situ following physiological experiments.</a> Cell Calcium. 21, 175-183 (1997).</li><li>Sagdullaev, B. T., Eggers, E. D., Purgert, R., Lukasiewicz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+interactions+between+excitatory+and+inhibitory+retinal+synapses+control+visual+output.">Nonlinear interactions between excitatory and inhibitory retinal synapses control visual output.</a> J. Neurosci. 31, 15102-15112 (2011).</li><li>Toychiev, A. H., Sagdullaev, B., Yee, C. W., Ivanova, E., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+time+and+cost+efficient+approach+to+functional+and+structural+assessment+of+living+neuronal+tissue.">A time and cost efficient approach to functional and structural assessment of living neuronal tissue.</a> J. Neurosci. Methods. 214, 105-112 (2013).</li><li>Yee, C. W., Toychiev, A. H., Sagdullaev, B. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Network+deficiency+exacerbates+impairment+in+a+mouse+model+of+retinal+degeneration.">Network deficiency exacerbates impairment in a mouse model of retinal degeneration.</a> Front Syst. Neurosci. 6, 8 (2012).</li><li>Ivanova, E., Muller, U., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+glycinergic+input+to+bipolar+cells+of+the+mouse+retina.">Characterization of the glycinergic input to bipolar cells of the mouse retina.</a> Eur. J. Neurosci. 23, 350-364 (2006).</li><li>Dmitrieva, N. A., Strang, C. E., Keyser, K. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+alpha+7+nicotinic+acetylcholine+receptors+by+bipolar,+amacrine,+and+ganglion+cells+of+the+rabbit+retina.">Expression of alpha 7 nicotinic acetylcholine receptors by bipolar, amacrine, and ganglion cells of the rabbit retina.</a> J. Histochem. Cytochem. 55, 461-476 (2007).</li></ol>

The hydrophilized PTFE membrane&#39;s high biocompatibility and transparency in solutions is advantageous when working with living tissue. This preparation has been successfully used in earlier work for patch-clamp recordings of light responses8-10. Here, we show how this approach can be adopted for structural analysis of the wholemount retina.
The bolus loading technique can be effectively used for labeling living tissue. This technique is aided by the structural support of the mem...

Performing immunohistochemistry in delicate intact tissues runs the risk of damage during handling and transfer. This can occur either in brain slices or other thin tissue, such as degenerated retina. Additionally, there are certain methods of tissue fixation that can be advantageous for immunostaining of neuronal structures, but result in compromised structural stability, precluding their use. A particular example of this is carbodiimide-based fixation, which is superior for staining receptors and hormones1-7 but is commonly avoided due to the instability of the fixed tissue.
Here, we describe a procedure which utilizes a hydrophilized PTFE membrane to structurally support delicate tissue, either fixed or unfixed, for a variety of staining techniques. The supporting membrane allows for the manipulation of delicate tissue before and after fixation, allowing for several steps of processing while minimizing the risk of tissue damage. Overall, this simple method to preserve tissue integrity provides the opportunity to use techniques that might otherwise be avoided. As such, this approach could also be successfully used for preparation of wide variety of tissues such as brain slices that become fragile following slicing procedures.

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="44">
			<td height="44" style="height:44px;width:260px;">Millicell Cell Culture Insert, 12 mm, 
			hydrophilic PTFE (Biopore), 0.4 &#956;m</td>
			<td style="width:183px;">Millipore</td>
			<td style="width:108px;">PICM01250</td>
			<td style="width:75px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Insulin syringe, 1 ml</td>
			<td>Beckton Dickinson</td>
			<td>309659</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Scissors</td>
			<td>Fine Science Tools</td>
			<td>15003-08</td>
			<td>dissection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Forceps, Dumont #55, inox</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>dissection</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cryostat</td>
			<td>Leica</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Confocal Microscope System</td>
			<td>Nikon</td>
			<td>various</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Capillary glass</td>
			<td>World Precision Instruments</td>
			<td>1B150F-4</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">P-97 Flaming/Brown Micropipette Puller</td>
			<td>Sutter Instrument Co.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Picospritzer III</td>
			<td>Parker Hannifin</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glass Bottom Culture Dishes</td>
			<td>MatTek Corporation</td>
			<td>P35G-0-14-C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Petri dish</td>
			<td>Falcon</td>
			<td>1008</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Disposable Graduated Transfer Pipettes</td>
			<td>VWR</td>
			<td>16001-180</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Multiwell plates, 24-well</td>
			<td>Beckton Dickinson</td>
			<td>351147</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cover glass, #1</td>
			<td>Electron Microscopy Sciences</td>
			<td>72200-30</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polysine adhesion slides</td>
			<td>Electron Microscopy Sciences</td>
			<td>63412-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microscope slides</td>
			<td>Globe</td>
			<td>1301</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Blocker</td>
			<td>Electron Microscopy Sciences</td>
			<td>71312</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">VECTASHIELD mounting medium</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">OCT medium</td>
			<td>Sakura</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Parafilm laboratory film</td>
			<td>Fisher</td>
			<td>13-374-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dow Corning high vacuum grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Mouse anti-PSD95</td>
			<td>Millipore</td>
			<td>MABN68</td>
			<td>antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Donkey anti-mouse Alexa 568</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td>antibody</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Isolectin Alexa 488</td>
			<td>Invitrogen</td>
			<td>I21411</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Chemiblocker</td>
			<td>Chemicon</td>
			<td>2170S</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sulforhodamine</td>
			<td>Sigma-Aldrich</td>
			<td>341738</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Carbodiimide</td>
			<td>Thermo Scientific</td>
			<td>22980</td>
			<td>EDC</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Demonstrated here are two representative experiments that benefit from the use of a hydrophilized PTFE membrane to support wholemount retina during staining procedures. The first experiment, illustrates the method for fast and simple characterization of retinal vasculature, an elaborate network of blood vessels that span across multiple retinal layers (Figure 2). This approach combines bolus loading of isolectin with immersion labeling with SRH. This is an efficient method of labeling living tissue that ...

Watch this Scientific Journal Video about Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry at JoVE.com

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

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 ...

optimized-protocol-for-retinal-wholemount-preparation-for-imaging

Research

JoVE Journal

Biology

22.8K vues.  Weill Medical College of Cornell University. 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.