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).</li>
	<li>Remove the cornea by cutting in a circular path along the ora serrata with small scissors, holding the eye at the limbus with forceps. Pull out the lens with forceps.</li>
	<li>Remove as much vitreous as possible, using blunted-edge forceps. This step is important for bolus injections and electrophysiological recordings; it is not necessary for when performing immunohistochemistry alone.</li>
	<li>Detach the retina from the eyecup by positioning forceps between the retina and the eyecup, moving the forceps slowly around the circumference. Stabilize the eye cup by holding the sclera firmly with a second set of forceps. Cut the optic nerve at the location between the retina and the eyecup to ease the separation of the retina from the eyecup.</li>
	<li>The retina can be dissected into pieces (e.g. quarters) or used as a whole. To cut into pieces, use scissors to make incisions from the retinal periphery all the way down to the optic nerve. Alternatively, to use the entire retina, make incisions from the periphery halfway to the optic nerve. The incisions will help to make a flat preparation.</li>
</ol>
2. Mounting onto Hydrophilized PTFE Membrane Insert
<ol>
	<li>Prepare the membrane as described below. When physiological recordings or dye injection experiments are performed, shorten the holder by using either a lathe or a handsaw so that the remaining ring has a height of 1 mm. 
	Note: The culture plate inserts may be either unmodified or modified (Figure 1A). The choice of the configuration is dictated by the purpose of the experiment. For immunostaining, an unmodified insert is suitable.</li>
	<li>Polish down rough surfaces with fine (300 grit or higher) sandpaper.</li>
	<li>(Optional) Remove the feet underneath the ring with scissors.</li>
	<li>To remove any debris, rinse or sonicate the membrane in distilled water for 2-3 min.</li>
	<li>Mount the tissue onto the membrane (Figure 1A). Place the tissue with some solution on top of the membrane with a plastic transfer pipette. Cut off the narrow tip of the pipette if necessary, to avoid having the tissue sample adhere to the pipette during transfer.</li>
	<li>Attach the retina with ganglion cells (GCs) up: on the membrane, gradually remove solution while spreading the retina and unfolding the folds with forceps.</li>
	<li>On the opposite side of the membrane, position an empty syringe (1 ml) directly underneath the tissue. Apply suction by pulling back on the plunger to a volume of 0.2-0.3 ml. Increase the surface area of suction by cutting the holding tip of the syringe prior to use. The tissue does not require further treatment for adhesion, as it is physically enmeshed within the membrane.</li>
</ol>
3. Bolus Injection of Isolectin Alexa 488
<ol>
	<li>Prepare patch pipettes and staining solution. Pull patch pipettes from borosilicate capillary glass with a Flaming/Brown puller, with a resistance of ~1 M&#8486;. Gently break the tip of the pipette.</li>
	<li>Make staining solution by adding 0.3 &#956;l of isolectin Alexa 488 stock solution to 10 &#956;l of the HEPES-buffered Ringer&#39;s solution described in step 1.2.</li>
	<li>Transfer sample to the patch-clamp setup for injection (Figure 1C). Place the membrane with attached tissue into the recording chamber perfused with warmed (37 &#176;C) and oxygenated HEPES-buffered Ringer&#39;s solution.</li>
	<li>Place the pipette containing dye against the inner limiting membrane (ILM, Figure 2A). Using micromanipulator, further advance the pipette against the ILM until it penetrates it. Inject the staining solution with a pressure 10-20 psi for 1 sec using a picospritzer. Single or multiple locations may be targeted for injection (Figure 2B). If a picospritzer is not available, a 10 ml syringe can be used to deliver a positive air pressure for injection.</li>
	<li>Incubate injected tissue for 10 min. During incubation, the injected tissue can be viewed under epifluorescence illumination.</li>
	<li>After labeling, the preparation may be taken to a microscope for imaging. Transfer the insert to a glass-bottom culture dish, placing retina side down (Figure 1C).</li>
	<li>Add a drop of HEPES solution to keep the specimen moist.</li>
	<li>Place 2-3 spots of vacuum grease around the perimeter of the plastic ring to firmly secure the insert to the bottom of the culture dish.</li>
	<li>Transfer the dish to the microscope stage.</li>
</ol>
4. Paraformaldehyde and Carbodiimide Fixation
<ol>
	<li>Fix the sample by transferring the insert into fixative for 15 min. Longer or shorter fixation times may be used, dependent on application. Both fixatives can be effectively used to fix GFP, isolectin, or fluorescent Alexa hydrazides (e.g. used for filling the cells in patch-clamp recordings). 
	Note: For fixation, either 4% carbodiimide or 4% paraformaldehyde solutions may be used. To maintain adherence, fixation should be done after the tissue has already been attached to the membrane. The tissue can be fixed immediately after mounting, or after physiological recording and/or bolus injection.</li>
	<li>Rinse the tissue in phosphate buffer (PB) for 30 min. After this, the tissue may be processed either as a wholemount (step 5) or cut into vertical sections and then processed (steps 6 and 7).</li>
</ol>
5. Immunostaining of Retinal Wholemounts
<ol>
	<li>In a 24-well dish (Figure 1D) block retinal pieces on the membrane for 1 hr in a staining solution.</li>
	<li>Dilute mouse anti-PSD-95 primary antibodies 1:300 in the staining solution and apply for 48 hr at room temperature in dark. Add ~100-150 &#956;l antibody solution in each well with the tissue. To prevent drying out, fill unused wells with distilled water.</li>
	<li>Rinse for 20 min in PB, 3x.</li>
	<li>Incubate for 24 hr in donkey anti-mouse Alexa 568 secondary antibody dissolved 1:500 in the staining solution.</li>
	<li>Rinse for 20 min in PB, 3x.</li>
	<li>Place the insert with the stained tissue into a standard 3 cm Petri dish filled with PB solution. With small scissors, cut out the portion of the membrane containing the retina from the ring.</li>
	<li>Transfer the retina on the membrane onto a slide retina up. Remove PB, add mounting medium. Place small pieces of a broken cover slip glass in between the slide and the coverslip glasses to avoid extensive squeezing and damage to the retina. Place coverslip.</li>
	<li>Seal the coverslip in place with nail polish. Keep slides in the dark, in a refrigerator.</li>
</ol>
6. Vertical Sectioning of Membrane-mounted Tissue
<ol>
	<li>After fixation, submerse the tissue on the membrane into the sucrose gradient solutions of 10%, 20%, and 30% sucrose for 30 min, 1 hr, and overnight, respectively. The overnight step is performed at 4 &#176;C in refrigerator, all other steps at room temperature.</li>
	<li>Using scissors, remove the membrane with the tissue from the plastic insert ring.</li>
	<li>Place the cut off membrane onto Parafilm with retina up and remove as much solution as possible with Kimwipes.</li>
	<li>Put the Parafilm with the tissue into the cryostat chamber and add a drop of OCT medium. Use the quick freezer device to accelerate the freezing.</li>
	<li>Detach the frozen sample and flip it over.</li>
	<li>Cut excess frozen OCT. Leave approximately 2 mm around the sample on three sides, and leave 5-6 mm from the side which will be attached to the holder.</li>
	<li>Cover exposed retinal side (membrane side) with a drop of OCT. Leave the attaching area free.</li>
	<li>Freeze the membrane with retinal tissue. Now the sample is ready for placing onto a cutting holder.</li>
	<li>Place a drop of OCT onto a cold holder ring. Immediately insert the frozen sample into the drop.</li>
	<li>Cut sections 10-20 &#956;m thick, setting cryostat temperature at -19 &#176;C. Use warm (room temperature) polysine adhesion slides. Leave the slides with the attached sections onto a heating platform for ~2-3 min (41 &#176;C) in dark to ensure firm adhesion of sections.</li>
	<li>Put the slides into a storage box and keep at -20 &#176;C until use.</li>
</ol>
7. Immunostaining of Retinal Sections
<ol>
	<li>Take the slides with the sections out of the freezer. Let them stay at room temperature for 1 min to remove the condensate.</li>
	<li>Encircle the sections with a liquid blocker to create a barrier for further incubation.</li>
	<li>Place the slides into the incubation box. Cover the bottom of the box with water to prevent evaporation of the antibody solution.</li>
	<li>Rinse with PB for 5 min.</li>
	<li>Prepare the same staining solutions and follow the steps as in step 5. The incubation with the primary antibody is overnight and with the secondary 1 hr. Do not use broken glass between the coverslip and the slide.</li>
</ol>

<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 authors have no competing financial interests.

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

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Biology

23.0K Views.  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.

Video: Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

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.

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.

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

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine parasitoids known to infect the host during the egg stage1. Second, it has a wide range of potential fruit fly hosts. Perhaps due to its life history, F. arisanus has been a successfully used for biological control of fruit flies in multiple tropical regions2-4. One impediment to the wide use of F. arisanus for fruit fly control is that it is difficult to establish a stable laboratory colony5-9. Despite this difficulty, in the 1990s USDA researchers developed a reliable method to maintain laboratory populations of F. arisanus10-12. There is significant interest in F. arisanus biology13,14, especially regarding its ability to colonize a wide variety of Tephritid hosts14-17; interest is especially driven by the alarming spread of Bactrocera fruit fly pests to new continents in the last decade18. Further research on F. arisanus and additional deployments of this species as a biological control agent will benefit from optimizations and improvements of rearing methods. In this protocol and associated video article we describe an optimized method for rearing F. arisanus based on a previously described approach12. The method we describe here allows rearing of F. arisanus in a small scale without the use of fruit, using materials available in tropical regions around the world and with relatively low manual labor requirements.

An Optimized Protocol for Rearing Fopius arisanus, a Parasitoid of Tephritid Fruit Flies

Fopius arisanus is an egg-larval parasitoid of Tephritid fruit flies that is successfully used in biological control of these important tropical pests. We describe here an optimized protocol for rearing F. arisanus on a small scale using readily available materials.

Fopius arisanus (Sonan) is an important parasitoid of Tephritid fruit flies for at least two reasons. First, it is the one of only three opiine ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has become the method of choice for introducing DNA into prokaryotes that are not naturally competent. Electroporation is a rapid, efficient, and streamlined transformation method that, in addition to purified DNA and competent bacteria, requires commercially available gene pulse controller and cuvettes. In contrast to the pulsing step, preparation of electrocompetent cells is time consuming and labor intensive involving repeated rounds of centrifugation and washes in decreasing volumes of sterile, cold water, or non-ionic buffers of large volumes of cultures grown to mid-logarithmic phase of growth. Time and effort can be saved by purchasing electrocompetent cells from commercial sources, but the selection is limited to commonly employed E. coli laboratory strains. We are hereby disseminating a rapid and efficient method for preparing electrocompetent E. coli, which has been in use by bacteriology laboratories for some time, can be adapted to V. cholerae and other prokaryotes. While we cannot ascertain whom to credit for developing the original technique, we are hereby making it available to the scientific community.

Rapid Protocol for Preparation of Electrocompetent Escherichia coli and Vibrio cholerae

Electroporation is a commonly employed method for introducing DNA into bacteria in a process known as transformation. Traditional protocols for the preparation of electrocompetent cells are time consuming and labor intensive. This article describes an alternate, rapid, and efficient method for the preparation of electrocompetent cells presently employed by some laboratories.

Electroporation has become a widely used method for rapidly and efficiently introducing foreign DNA into a wide range of cells. Electrotransformation has ...

Detection of Modified Forms of Cytosine Using Sensitive Immunohistochemistry

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. 5-hydroxylmethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are the recently discovered modified cytosine bases produced by enzymatic oxidation of 5mC, whose biological functions remain relatively obscure. A number of approaches ranging from biochemical to antibody based techniques have been employed to study the genomic distribution and global content of these modifications in various biological systems. Although some of these approaches can be useful for quantitative assessment of these modified forms of 5mC, most of these methods do not provide any spatial information regarding the distribution of these DNA modifications in different cell types, required for correct understanding of their functional roles. Here we present a highly sensitive method for immunochemical detection of the modified forms of cytosine. This method permits co-detection of these epigenetic marks with protein lineage markers and can be employed to study their nuclear localization, thus, contributing to deciphering their potential biological roles in different experimental contexts.

Herein we describe a sensitive immunochemical method for mapping the spatial distribution of 5mC oxidation derivatives based on the use of peroxidase-conjugated secondary antibodies and tyramide signal amplification.

Methylation of cytosine bases (5-methylcytosine, 5mC) occurring in vertebrate genomes is usually associated with transcriptional silencing. ...

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...

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 ...