We are grateful to Dr. Sachiko Sato and Julie-Christine L&#233;vesque at the Bioimaging Platform of the Centre de recherche du CHU de Qu&#233;bec for their technical assistance. Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) RGPIN-2014-05308, The Banting Research Foundation, and The Scottish Rite Charitable Foundation of Canada to M.E.T supported this work.
H.E.H. is recipient of a scholarship from the Lebanese Ministry of Education and Higher Education, and K.B. from the Facult&#233; de m&#233;decine of Universit&#233; Laval.

Note: All experiments were approved and performed under the guidelines of the Institutional animal ethics committee, in conformity with the Canadian Council on Animal Care guidelines as administered by the Animal Care Committee of Universit&#233; Laval. APP-PS1 male mice between 4 and 21 months of age were used. These animals were housed under a 12&#160;hr light-dark cycle at 22 - 25 &#176;C with free access to food and water.
1. Methoxy-X04 Solution Preparation
<ol>
	<li>Prepare a 5 mg/ml solution of methoxy-X049 by dissolving methoxy-X04 into a solution containing 10% dimethyl sulfoxide (DMSO), 45% propylene glycol, and 45% sodium phosphate buffered saline (0.9% NaCl in 100 mM phosphate buffer, pH 7.4).
	<ol>
		<li>Using a microbalance, weigh 5 mg of the methoxy-X04 compound. Under a fume hood, dissolve the methoxy-X04 in DMSO and stir until a clear greenish solution is obtained. Successively add propylene glycol and phosphate buffer saline while stirring with each addition.</li>
		<li>Stir the solution at 4&#160;&#176;C on a rotator O/N. Obtain a yellowish green emulsion. The solution may be stored at 4&#160;&#176;C for up to two months without degradation.</li>
	</ol>
	</li>
</ol>
2. Methoxy-X04 Solution Injection
<ol>
	<li>24 hr&#160;prior to perfusion, weigh the mice and give each mouse an intraperitoneal injection of methoxy-X04 at a dose of 10 mg/kg of body weight using a 27 &#189; G&#160;needle.</li>
</ol>
3. Transcardial Perfusion of the Injected Mice
<ol>
	<li>On the day before the perfusion, prepare appropriate volumes of phosphate buffered saline (PBS), 3.5% acrolein, and 4% paraformaldehyde (PFA) solutions13 and store them at 4 &#176;C O/N. 
	Note: These solutions will be used for both perfusion and pre-embedding immunohistochemistry. Take special care when working with acrolein, as&#160;it is corrosive and toxic to both researchers and the environment. In addition, since acrolein is corrosive to plastic, always use suitable glassware to prepare acrolein solutions.</li>
	<li>On the day of perfusion, filter the PFA and acrolein by using coarse filter paper of 25 &#181;m particle retention.</li>
	<li>During the perfusion, use a peristaltic pump to deliver 15 ml of PBS, 75 ml of acrolein, and 150 ml of PFA successively into the mouse circulation, at a flow rate of 25 ml/min. 
	Use caution.&#160;Perform perfusion strictly inside a fume hood to avoid harmful fumes of PFA and acrolein.
	<ol>
		<li>To set&#160;up the pump, insert one end into the PBS solution, fill the tubing (holding approximately 15 ml) with PBS, and fix a 25 G&#160;winged blood collection needle to the other end. At all times, ensure that the tubing is free from any trapped air bubbles.</li>
		<li>Place the tube directly in the acrolein bottle after setting the perfusion pump with the tubing filled with PBS.</li>
	</ol>
	</li>
	<li>Anaesthetize one mouse at a time with a 90 mg/kg of body weight dose of sodium pentobarbital injected intraperitoneally using a 27 &#189;&#160;G needle. Assess responses to tail/toe pinches. Proceed only if the mouse is unresponsive to such aversive, painful stimuli.</li>
	<li>Secure the mouse in the supine position (lying on the back with face upward) by taping the forepaws and hindpaws to the work surface and carefully expose the heart without causing damage to other organs. Be sure to work quickly after puncturing the diaphragm.
	<ol>
		<li>Perform an incision through the skin with surgical scissors along the thoracic midline beginning caudal to the ribcage and proceed rostral to the clavicle.</li>
		<li>Make two additional lateral incisions along the base of the ventral ribcage. Gently move and pin the two flaps of skin, making sure to expose the entire thoracic cavity.</li>
		<li>Hold the xiphoid process with blunt forceps to expose the thoracic cavity and steady the pointed scissors. Cut through the diaphragm and ribcage, being careful to avoid puncturing the lungs, and continue the incision rostral to the clavicles. Be sure to work quickly after puncturing the diaphragm.</li>
	</ol>
	</li>
	<li>Gently tear the pericardial sac with blunt forceps. Hold the heart steady with blunt forceps, cut the right atrium, and start the infusion of PBS. Immediately insert the blood collection needle into the left ventricle.</li>
	<li>Perfuse the mouse with PBS (in the tubing) followed by acrolein for 3 min&#160;(corresponding to 75 ml) then switch to PFA for 6 min&#160;(corresponding to 150 ml). Be sure to pause the pump flow before switching solution to prevent bubbles from entering the tube.</li>
	<li>Decapitate the mouse and extract the fixed brain and place it directly into a glass vial containing 4% PFA for at least 2 hr&#160;at 4 &#176;C.
	<ol>
		<li>To extract the brain, use scissors and tweezers to cut through the skin to expose the skull. Carefully break the skull open in between the eyes, and chip off small pieces of it to expose the underlying brain.</li>
		<li>Carefully remove the exposed brain and post&#160;fix it with 4% PFA for an additional 2 hr&#160;before proceeding for vibratome sectioning.</li>
	</ol>
	</li>
</ol>
4. Brain Sectioning Using a Vibratome
<ol>
	<li>Wash the fixed brain 3 times with chilled PBS. Using a sharp razor blade, remove the olfactory bulb (unless this region is under investigation) and cut the brain transversally into 2 - 3 pieces of approximately equal height, all of which can be sectioned simultaneously in order to accelerate the procedure.</li>
	<li>Glue the pieces of brain tissue vertically onto the specimen plate secured into the tray. Make sure that the smooth cut surface sticks firmly to the specimen plate and does not get dislodged during the sectioning procedure. Add enough PBS solution into the tray until the entire brain surface is completely submerged. It is important to keep the brain pieces and subsequent sections fully immersed in PBS throughout this step.</li>
	<li>Place the tray in the vibratome, adjust the sectioning speed to 0.5 mm/sec, the sectioning frequency to 90 Hz, and the feed thickness to 50 &#181;m in order to yield 50 &#181;m thick sections. Then transfer the sections into 20 ml glass vials containing cryoprotectant solution (40% PBS, 30% ethylene glycol, and 30% glycerol) using a fine paintbrush. Store the vials at -20 &#176;C until further use. Alternatively, keep the sections in PBS for immediate screening as described in the following steps.</li>
</ol>
5. Section Screening for the Presence of Methoxy-X04-stained Plaques
<ol>
	<li>With the aid of a stereotaxic mouse brain atlas14, select brain sections containing the region of interest, for instance, the hippocampus CA1 as used in the present example. Place each section into a well within a 24-well culture plate containing enough cryoprotectant solution so as to prevent the sections from drying out.</li>
	<li>Examine each section successively, to avoid drying out the sections, using the following procedure:
	<ol>
		<li>Add a droplet of PBS to a microscope slide with a disposable pipette, and place the section on the droplet.</li>
		<li>Examine the section under a fluorescent microscope to identify regions containing methoxy-X04 labeled A&#946; plaques. 
		Note: methoxy-X04 can be easily visualized using a fluorescence ultraviolet (UV) filter (excitation 340 - 380 nm).</li>
	</ol>
	</li>
	<li>Capture images of regions of interest (ROI) in bright field as well as in fluorescence mode without moving the microscope stage, since each image must show the same region in both fields in order to correlate the presence of the plaques directly to the structural region of the tissue section.</li>
	<li>Save and name the images taken according to the animal number.&#160;Also record the well number in the plate and the field of the pictures taken. For example, Ex: 9978A1B; Animal number: 9978; Well number: A1; Field: Bright. Place the section back in&#160;its designated well once the imaging is completed. 
	Note: In the end, for each section examined, two pictures are obtained, one in bright field and another in UV field.</li>
	<li>Open the two images of the same ROI (one in bright field and the other in UV field) in Image J, and using the MosaicJ plugin, align the edges of the two images and save the aligned and combined image according to the well number it came from.</li>
	<li>Save the picture in a separate folder with the number of the particular animal. Combine the images to identify and localize the plaques in the tissue section, and have a full view of the whole section in the two different fields (bright and UV).</li>
	<li>After the screening process is completed, store the examined sections at -20&#160;&#176;C in a 24-well culture plate containing cryoprotectant until immunostaining or further processing is carried out.</li>
</ol>
6. Pre-embedding Immunostaining for IBA1
Note: Perform the immunostaining for IBA1 on selected freely-floating sections by placing the plate on a slowly moving rocker at RT.
<ol>
	<li>Thoroughly wash the sections 3 times with approximately 1 ml PBS, each wash lasting for 10 min.</li>
	<li>Quench endogenous peroxidases with 0.3% hydrogen peroxide in PBS solution for 10 min. This step prevents non-specific peroxidase activity, which is important to avoid background staining.</li>
	<li>Wash the tissue in PBS 3 times for 10 min&#160;each.</li>
	<li>Incubate the sections in 0.1% sodium borohydride in PBS solution for 30 min. This step is important to reduce any remaining aldehydes from the fixation step, and is especially important if acrolein is used in tissue fixation.</li>
	<li>Wash the tissue in PBS 3 times for 10 min&#160;each time and make sure to remove all the bubbles.</li>
	<li>Block the sections for 1 hr. Different antibodies may require different blocking times and solutions. For IBA1, prepare blocking buffer containing 10% fetal bovine serum, 3% bovine serum albumin, and 0.01% Triton X-100 in 50 mM Tris-buffered saline (TBS; pH 7.4). 
	Note: The blocking step is to prevent nonspecific binding of the primary antibody. The low concentration of Triton X-100 allows slight permeabilization of membranes for better staining, and is low enough to preserve most ultrastructural features of tissue under EM.</li>
	<li>Remove the blocking buffer from the tissue, and incubate in primary antibody solution (rabbit anti-IBA1, diluted [1:1,000] in blocking buffer) at 4 &#176;C O/N.</li>
	<li>Wash in TBS 3 times for 10 min&#160;each time.</li>
	<li>Incubate in secondary antibody (goat anti-rabbit conjugated to biotin) [1:200] in 0.05 M TBS for 90 min.</li>
	<li>Wash in TBS 5 times for 5 min&#160;each time.</li>
	<li>Incubate in Avidin-Biotin complex solution (Avidin [1:100], Biotin [1:100]) in TBS solution for 1 hr.</li>
	<li>Wash in TBS 5 times for 5 min&#160;each time.</li>
	<li>Reveal the IBA1 staining with 0.05% diaminobenzidine (DAB) and 0.015% hydrogen peroxide in TBS for 8 min. 
	Note: The timing of DAB development may vary from protocol to protocol and should be determined by quickly examining the stained sections under a light microscope. 
	Note: For IBA1, one should be able to clearly visualize the cell bodies and processes of the IBA1-stained microglia at 20X (see Figure 2 for representative example). Be cautious using DAB, as it is a known carcinogen.</li>
	<li>Avoid over-developing by carefully monitoring the sections (as described above) during this step. Stop the reaction by washing the sections in chilled PB 5 times for 5 min&#160;each time.</li>
</ol>
7. Processing for Electron Microscopy
<ol>
	<li>Prepare 1% osmium tetroxide solution in PBS in a glass vial. Osmium tetroxide is photosensitive;&#160;cover the glass vial with aluminum foil to protect the solution from light. 
	Be cautious using osmium tetroxide, as it is a very hazardous chemical. Perform this and the following steps inside a fume hood.</li>
	<li>Remove the PBS from the sections and spread them flat using a fine paintbrush. Perform this step one well at a time to keep&#160;the sections from drying out. Be sure to flatten the sections immediately before adding osmium tetroxide, as any folds in the sections will become permanent and attempting to flatten tissue post osmium fixation will only break the sections.</li>
	<li>Immerse the sections in osmium tetroxide for 30 min&#160;at RT, adding one drop of osmium tetroxide at a time with a transfer pipette, to prevent the sections from folding. Cover the well with aluminum foil to protect the sections from light. 
	Note: This step fixes the lipids within the sections. The tissue will appear very dark after osmium post-fixation.</li>
	<li>While sections are in osmium tetroxide, prepare plastic resin in a disposable beaker by mixing 20 g component A, 20 g component B, 0.6 g component C, and 0.4 g component D. Combine the components together in the above order and mix them well using a 10 ml serological pipette until a uniform color is obtained.</li>
	<li>Transfer the prepared mixture to aluminum weighing dishes. They will receive the tissue sections once they have been dehydrated.</li>
	<li>Dehydrate the sections in increasing concentrations of ethanol for 2 min in the following order: 35%, 35%, 50%, 70%, 80%, 90%, 100%, 100%, 100%.</li>
	<li>To remove residual ethanol, immerse the sections in propylene oxide 3 times for 2 min each time. Remove the dehydrated-sections from the 24-well culture plate into 20 ml glass vials. Always use glass vials when working with propylene oxide as it dissolves plastic, and maintain caution, as it is hazardous.</li>
	<li>Use a bent glass pipette tip or a fine paintbrush to transfer the sections from propylene oxide solution into the resin and leave O/N for infiltration at RT. Be careful not to dilute the resin with propylene oxide.</li>
	<li>Embed the section with the resin on poly-chloro-tri-fluoro-ethylene (PCTFE) filmsheets:
	<ol>
		<li>Place the aluminum weighing dishes containing the specimens into a 50 - 60 &#176;C oven for 10 - 15 min&#160;prior to embedding the sections on PCTFE filmsheets.</li>
		<li>When embedding sections, work with one aluminum weighing dish at a time. Using a fine paintbrush, paint a thin layer of resin onto one PCTFE film sheet.</li>
		<li>Move one section of tissue at a time from the aluminum weighing dish to the PCTFE film sheet. Remove excess resin from around the tissue, being careful not to disturb it.</li>
		<li>After moving all the sections from one weighing dish to the PCTFE film sheet, place a second PCTFE sheet over the first, creating a sandwich of tissue and resin in between 2 sheets.</li>
	</ol>
	</li>
	<li>Polymerize the resin in an incubator for 3 days at 55 - 60 &#176;C.</li>
	<li>The material is now ready for ultrathin sectioning and ultrastructural examination, specialized techniques which are often performed by EM core facilities. Store the samples between PCTFE film sheets safely at RT without ultrastructural degradation. 
	Note: The subsequent steps for ultrathin sectioning and transmission electron microscopy examination are explained in a separate protocol13.</li>
</ol>

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The authors have nothing to disclose.

This protocol explains a correlative approach for targeting dense core A&#946; plaques with EM. Methoxy-X04 in vivo injection allows rapid selection of brain sections that contain A&#946; plaques within particular regions and layers of interest, for instance the hippocampus CA1, strata radiatum, and lacunosum-moleculare. In the present example, methoxy-X04 pre-screening was combined with pre-embedding immunostaining for IBA1 to study how different microglial phenotypes interact with synapses at the ultrastructur...

Amyloid A&#946; plaque formation is the main neuropathological hallmark of Alzheimer&#39;s disease (AD). However increasing evidence suggests important roles of the immune system in disease progression1,2. In particular, new data from preclinical and clinical studies established immune dysfunction as a main driver and contributor to AD pathology. With these findings, central and peripheral immune cells have emerged as promising therapeutic targets for AD3. The following protocol combines light and electron microscopy (EM) to generate new insights into the relationship between A&#946; plaque deposition and microglial phenotypic alterations in AD. This protocol allows the labeling of A&#946; plaques in mouse models of AD using in vivo injection of the fluorescent dye methoxy-X04. Methoxy-X04 is a Congo Red derivative that can easily cross the blood-brain barrier to enter the brain parenchyma and bind &#946;-pleated sheets with high affinity. Since the dye is fluorescent, it can be used for in vivo detection of A&#946; plaque deposition with two-photon microscopy4. Once bound to A&#946;, methoxy-X04 does not dissociate or redistribute away from plaques, and it retains its fluorescence over time. It is generally administered peripherally to allow for non-invasive imaging of brain dynamics5. The fluorescence also remains following aldehyde fixation, allowing for correlative post-mortem analyses, including investigation of neuronal death in the vicinity of A&#946; plaques6.
This protocol takes advantage of the properties of methoxy-X04 to select brain sections from APPSWE/PS1A246E mice (APP-PS1; coexpressing a double mutation at APP gene Lys670Asn/Met671Leu, and human presenilin PS1-A264E variant)7 that exhibit A&#946; plaques in specific regions of interest (hippocampus CA1, strata radiatum, and lacunosum-moleculare) prior to pre-embedding immunostaining against the microglial marker ionized calcium-binding adapter molecule 1 (IBA1) to visualize microglial cell bodies and processes with EM. The mice are given intraperitoneal injection of methoxy-X04 solution, 24 hr&#160;prior to brain fixation through transcardial perfusion. Coronal brain sections are obtained using a vibratome. Sections containing the hippocampus CA1 are screened under a fluorescent microscope for the presence of A&#946; plaques in strata radiatum and lacunosum-moleculare. Immunostaining for IBA1, osmium tetroxide post-fixation, and plastic resin embedding are then performed on the selected brain sections. At the end of this protocol, the sections can be archived without further ultrastructural degradation, ready for ultrathin sectioning and ultrastructural examination. Importantly, the plaques are still fluorescent after immunostaining with different antibodies, for instance IBA1 as in the present protocol. They become darker than their surrounding neuropil following osmium tetroxide post-fixation, independently of methoxy-X04 staining, which helps to accurately identify the regions of interest, generally down to a few square millimeters, to be examined with the transmission electron microscope.
This correlative approach offers an efficient way to identify specific brain sections to examine at the ultrastructural level. This is particularly helpful when studying early AD pathology, within specific brain regions or layers that may only contain a few A&#946; plaques, present in only a small fraction of tissue sections. During these times especially, it would be inefficient to use immunostaining for A&#946; (and dual labeling for other cellular markers such as IBA1) on several brain sections simply to yield a small fraction containing A&#946; plaques at the right location. In addition, injection of live mice with methoxy-X04 prior to sacrifice and tissue processing does not compromise the ultrastructural preservation. Alternative methods such as post-mortem staining with Congo Red, Thioflavin S, Thioflavin T or methoxy-X04 on fixed tissue sections require staining differentiation in ethanol,8-11 which causes osmotic stress and disrupts the ultrastructure. Congo Red is also a known human carcinogen12.

<table border="0" cellpadding="0" cellspacing="0" width="1006">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Methoxy-X04</td>
			<td style="width:197px;">Tocris Bioscience</td>
			<td style="width:119px;">4920</td>
			<td style="width:167px;">10 mg substrate per tablet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene glycol</td>
			<td>Sigma Aldrich</td>
			<td>W294004&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher BioReagents</td>
			<td>BP231-1</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride NaCl</td>
			<td>Sigma</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate monobasic monohydrate</td>
			<td>Sigma</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td>Sigma</td>
			<td>S0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris Hydrochloride</td>
			<td>Fisher BioReagents</td>
			<td>BP153500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Acrolein&#160;</td>
			<td>Sigma</td>
			<td>110221</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Granular</td>
			<td>Electron Microscopy Sciences</td>
			<td>19210</td>
			<td>Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter paper</td>
			<td>Fisher</td>
			<td>09-790-14F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peristaltic Pump with Tubing</td>
			<td>ColeParmer</td>
			<td>cp.78023-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Excel Winged blood Collection Set Needles 25G&#160;</td>
			<td>Becton Dickinson</td>
			<td>367341</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Extrafine Forceps</td>
			<td>F.S.T</td>
			<td>11152-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scissors</td>
			<td>F.S.T</td>
			<td>14090-09</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hartman Hemostats</td>
			<td>F.S.T</td>
			<td>13003-10</td>
			<td>Tip shape: curved</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical Scissors</td>
			<td>F.S.T</td>
			<td>14004-16</td>
			<td>Tip shape: straight</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micro Dissecting Scissors</td>
			<td>ROBOZ surgical store</td>
			<td>5818</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass scintillation vials</td>
			<td>Fisher Scientific</td>
			<td>74515-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibrating Blade Michrotome Leica VT1000 S</td>
			<td>Leica Biosystems&#160;</td>
			<td>14047235612</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome blades</td>
			<td>Electron microscopy Sciences</td>
			<td>71990</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24-well Tissue Culture Plates</td>
			<td>Fisher Scientific</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylene Glycol</td>
			<td>Fisher BioReagents</td>
			<td>BP230-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Fisher BioReagents</td>
			<td>BP229-4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide, 30%</td>
			<td>J.T.BAKER</td>
			<td>cat: 2186-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium borohydride</td>
			<td>Sigma</td>
			<td>480886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris HCl</td>
			<td>Fisher</td>
			<td>BP153-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine Serum (FBS)</td>
			<td>Sigma Aldrich</td>
			<td>F1051</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA), fraction V</td>
			<td>Thomas Scientific</td>
			<td>C001H24</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti IBA1, Rabbit&#160;</td>
			<td>WAKO</td>
			<td>019-19741</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Goat Anti-Rabbit IgG</td>
			<td>Jackson Immunoresearch</td>
			<td>111066046</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VECTASTAIN Elite ABC Kit (Standard)</td>
			<td>Vector Labs</td>
			<td>PK-6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3.3&#39;-Diaminobenzidine tetra-hydrochloride (DAB)</td>
			<td>Sigma</td>
			<td>D5905-50TAB</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Osmium tetroxide, 4% solution</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td>Caution: toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component A</td>
			<td>Sigma</td>
			<td>44611</td>
			<td>Resin Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component B</td>
			<td>Sigma</td>
			<td>44612</td>
			<td>Hardener Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component C</td>
			<td>Sigma</td>
			<td>44613</td>
			<td>Plasticizer Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Durcupan&#8482; ACM single component D</td>
			<td>Sigma</td>
			<td>44614</td>
			<td>Accelerator Caution: Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol</td>
			<td>LesAlcoolsdeComerce</td>
			<td>151-01-15N</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Propylene oxide</td>
			<td>Sigma</td>
			<td>110205</td>
			<td>Caution: corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum weigh dishes</td>
			<td>Electron Microscopy Sciences</td>
			<td>70048-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ACLAR&#174;&#8211;Fluoropolymer Films</td>
			<td>Electron Microscopy Sciences</td>
			<td>50425</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oven/Incubator</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label &#946;-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for A&#946; (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing A&#946; plaques at the right location. Importantly, A&#946; plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope.

This section illustrates the results that can be obtained at different critical steps of the protocol. In particular the results show examples of brain sections containing methoxy-X04 stained plaques in specific region and layers of interest: the hippocampus CA1, strata radiatum, and lacunosum-moleculare. The plaques and regional/lamellar organization of the hippocampus are successively visualized using a combination of UV and bright field filters (Figure 1). The selected...

Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with ÃŽÂ²-Amyloid Plaques at JoVE.com

Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques

This article describes a protocol for visualizing amyloid A&#946; plaques in Alzheimer&#39;s disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to &#946;-pleated sheets found in dense core A&#946; plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

A detailed protocol is provided here to identify amyloid A&#946; plaques in brain sections from Alzheimer&#39;s disease mouse models before pre-embedding ...

correlative-light-electron-microscopy-to-study-microglial

Research

JoVE Journal

Neuroscience

11.2K Views.  CHU de Québec Research Center. This article describes a protocol for visualizing amyloid Aβ plaques in Alzheimer's disease mouse models using methoxy-X04, which crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. It allows pre-screening of plaque-containing tissue sections prior to immunostaining and processing for electron microscopy.

Video: Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques

Focussed Ion Beam Milling and Scanning Electron Microscopy of Brain Tissue

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron microscope (FIB/SEM). The samples are fixed with aldehydes, heavy metal stained using osmium tetroxide and uranyl acetate. They are then dehydrated with alcohol and infiltrated with resin, which is then hardened. Using a light microscope and ultramicrotome with glass knives, a small block containing the region interest close to the surface is made. The block is then placed inside the FIB/SEM, and the ion beam used to roughly mill a vertical face along one side of the block, close to this region. Using backscattered electrons to image the underlying structures, a smaller face is then milled with a finer ion beam and the surface scrutinised more closely to determine the exact area of the face to be imaged and milled. The parameters of the microscope are then set so that the face is repeatedly milled and imaged so that serial images are collected through a volume of the block. The image stack will typically contain isotropic voxels with dimenions as small a 4 nm in each direction. This image quality in any imaging plane enables the user to analyse cell ultrastructure at any viewing angle within the image stack.

This protocol describes how resin embedded brain tissue can be prepared and imaged in the three dimensions in the focussed ion beam, scanning electron microscope.

This protocol describes how biological samples, like brain tissue, can be imaged in three dimensions using the focussed ion beam/scanning electron ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Micron-scale Resolution Optical Tomography of Entire Mouse Brains with Confocal Light Sheet Microscopy

Understanding the architecture of mammalian brain at single-cell resolution is one of the key issues of neuroscience. However, mapping neuronal soma and projections throughout the whole brain is still challenging for imaging and data management technologies. Indeed, macroscopic volumes need to be reconstructed with high resolution and contrast in a reasonable time, producing datasets in the TeraByte range. We recently demonstrated an optical method (confocal light sheet microscopy, CLSM) capable of obtaining micron-scale reconstruction of entire mouse brains labeled with enhanced green fluorescent protein (EGFP). Combining light sheet illumination and confocal detection, CLSM allows deep imaging inside macroscopic cleared specimens with high contrast and speed. Here we describe the complete experimental pipeline to obtain comprehensive and human-readable images of entire mouse brains labeled with fluorescent proteins. The clearing and the mounting procedures are described, together with the steps to perform an optical tomography on its whole volume by acquiring many parallel adjacent stacks. We showed the usage of open-source custom-made software tools enabling stitching of the multiple stacks and multi-resolution data navigation. Finally, we illustrated some example of brain maps: the cerebellum from an L7-GFP transgenic mouse, in which all Purkinje cells are selectively labeled, and the whole brain from a thy1-GFP-M mouse, characterized by a random sparse neuronal labeling.

In this article we describe the full experimental procedure to reconstruct, with high resolution, the fine brain anatomy of fluorescently labeled mouse brains. The described protocol includes sample preparation and clearing, specimen mounting for imaging, data post-processing and multi-scale visualization.

Understanding the architecture of mammalian  brain at single-cell resolution is one of the key issues of neuroscience.  However, mapping neuronal soma and ...

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow for an in-depth characterization to identify pathways involved in these events. Cerebellar granule neurons (CGNs) that are derived from the developing cerebellum are an ideal model system that allows for morphological analyses. Here, we describe a method of how to genetically manipulate CGNs and how to study axono- and dendritogenesis of individual neurons. With this method the effects of RNA interference, overexpression or small molecules can be compared to control neurons. In addition, the rodent cerebellar cortex is an easily accessible in vivo system owing to its predominant postnatal development. We also present an in vivo electroporation technique to genetically manipulate the developing cerebella and describe subsequent cerebellar analyses to assess neuronal morphology and migration.

Genetic Manipulation of Cerebellar Granule Neurons In Vitro and In Vivo to Study Neuronal Morphology and Migration

Neuronal morphogenesis and migration are crucial events underlying proper brain development. Here, we describe methods to genetically manipulate cultured cerebellar granule neurons and the developing cerebellum for the assessment of morphology and migratory characteristics of neurons.

Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of the olfactory epithelium continuously give rise to new sensory neurons that extend their axons into the olfactory bulb, where they face the challenge to integrate into existing circuitry. Because of this particular feature, the olfactory system represents a unique opportunity to monitor axonal wiring and guidance, and to investigate synapse formation. Here we describe a procedure for in vivo labeling of sensory neurons and subsequent visualization of axons in the olfactory system of larvae of the amphibian Xenopus laevis. To stain sensory neurons in the olfactory organ we adopt the electroporation technique. In vivo electroporation is an established technique for delivering fluorophore-coupled dextrans or other macromolecules into living cells. Stained sensory neurons and their axonal processes can then be monitored in the living animal either using confocal laser-scanning or multiphoton microscopy. By reducing the number of labeled cells to few or single cells per animal, single axons can be tracked into the olfactory bulb and their morphological changes can be monitored over weeks by conducting series of in vivo time lapse imaging experiments. While the described protocol exemplifies the labeling and monitoring of olfactory sensory neurons, it can also be adopted to other cell types within the olfactory and other systems.

The Olfactory System as a Model to Study Axonal Growth Patterns and Morphology In Vivo

We describe a protocol for in vivo labeling of olfactory sensory neurons by electroporation and subsequent confocal laser-scanning or multiphoton microscopy to visualize neuronal morphology and its development over time.

The olfactory system has the unusual capacity to generate new neurons throughout the lifetime of an organism. Olfactory stem cells in the basal portion of ...

A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the barrier and the brain parenchyma is still an unmet need. To fill this gap, we designed a 2D in cellulo model, the BBB-Minibrain, by combining a polyester porous membrane culture insert human BBB model with a Minibrain formed by a tri-culture of human brain cells (neurons, astrocytes and microglial cells). The BBB-Minibrain allowed us to test the transport of a neuroprotective drug candidate (e.g., Neurovita), through the BBB, to determine the specific targeting of this molecule to neurons and to show that the neuroprotective property of the drug was preserved after the drug had crossed the BBB. We have also demonstrated that BBB-Minibrain constitutes an interesting model to detect the passage of virus particles across the endothelial cells barrier and to monitor the infection of the Minibrain by neuroinvasive virus particles. The BBB-Minibrain is a reliable system, easy to handle for researcher trained in cell culture technology and predictive of the brain cells phenotypes after treatment or insult. The interest of such in cellulo testing would be twofold: introducing derisking steps early in the drug development on the one hand and reducing the use of animal testing on the other hand.

Here we present a protocol describing the setting of an in cellulo BBB (Blood brain barrier)-Minibrain polyester porous membrane culture insert system in order to evaluate the transport of biomolecules or infectious agents across a human BBB and their physiological impact on the neighboring brain cells.

The early screening of nervous system medicines on a pertinent and reliable in cellulo BBB model for their penetration and their interaction with the ...

Light-Induced Molecular Adsorption of Proteins Using the PRIMO System for Micro-Patterning to Study Cell Responses to Extracellular Matrix Proteins

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the cells themselves. Here, we present the method of Light-Induced Molecular Adsorption of Proteins (LIMAP) using the PRIMO system as a patterning technique to produce micro-patterned extracellular matrix (ECM) substrates using a single or combination of proteins. The method enables printing of ECM patterns in micron resolution with excellent reproducibility. We provide a step-by-step protocol and demonstrate how this can be applied to study the processes of neuronal pathfinding. LIMAP has significant advantages over existing micro-printing methods in terms of the ease of patterning more than one component and the ability to generate a pattern with any geometry or gradient. The protocol can easily be adapted to study the contribution of almost any chemical component towards cell fate and cell behavior. Finally, we discuss common issues that can arise and how these can be avoided.

Our overall aim is to understand how cells sense extracellular cues that lead to directed axonal growth. Here, we describe the methodology of Light-Induced Molecular Adsorption of Proteins, used to produce defined micro-patterns of extracellular matrix components in order to study specific events that govern axon outgrowth and pathfinding.

Cells sense a variety of extracellular cues, including the composition and geometry of the extracellular matrix, which is synthesized and remodeled by the ...

Serial Block-Face Scanning Electron Microscopy (SBEM) for the Study of Dendritic Spines

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In addition, some features of dendritic spines, such as volume of the spine and post-synaptic density (PSD) (representing post-synaptic part of the synapse), presence of presynaptic terminal, and smooth endoplasmic reticulum or atypical form of PSD (e.g., multi-innervated spines), can be observed only with 3D EM. By employing serial block-face scanning electron microscopy (SBEM) it is possible to obtain 3D EM data easier and in a more reproducible manner than when performing traditional serial sectioning. Here we show how to prepare mouse hippocampal samples for SBEM analysis and how this protocol can be combined with immunofluorescence study of dendritic spines. Mild fixation perfusion allows us to perform immunofluorescence studies with light microscopy on one half of the brain, while the other half was prepared for SBEM. This approach reduces the number of animals to be used for the study.

Serial Block-Face Scanning Electron Microscopy SBEM for the Study of Dendritic Spines

Serial Block-Face Scanning Electron Microscopy (SBEM) is applied to image and analyze dendritic spines in the murine hippocampus.

Three-dimensional electron microscopy (3D EM) gives a possibility to analyze morphological parameters of dendritic spines with nanoscale resolution. In ...

Primary Cultures of Rat Astrocytes and Microglia and Their Use in the Study of Amyotrophic Lateral Sclerosis

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to use these cells for the purpose of studying the pathophysiology of amyotrophic lateral sclerosis (ALS) in the rat hSOD1G93A model. First, the protocol shows how to isolate and culture astrocytes and microglia from postnatal rat cortices, and then how to characterize and test these cultures for purity by immunocytochemistry using the glial fibrillary acidic protein (GFAP) marker of astrocytes and the ionized calcium-binding adaptor molecule 1 (Iba1) microglial marker. In the next stage, methods are described for dye-loading (calcium-sensitive Fluo 4-AM) of cultured cells and the recordings of Ca2+ changes in video imaging experiments on live cells.
The examples of video recordings consist of: (1) cases of Ca2+ imaging of cultured astrocytes acutely exposed to immunoglobulin G (IgG) isolated from ALS patients, showing a characteristic and specific response compared to the response to ATP as demonstrated in the same experiment. Examples also show a more pronounced transient rise in intracellular calcium concentration evoked by ALS IgG in hSOD1G93A astrocytes compared to non-transgenic controls; (2) Ca2+ imaging of cultured astrocytes during a depletion of calcium stores by thapsigargin (Thg), a non-competitive inhibitor of the endoplasmic reticulum Ca2+ ATPase, followed by store-operated calcium entry elicited by the addition of calcium in the recording solution, which demonstrates the difference between Ca2+ store operation in hSOD1G93A and in non-transgenic astrocytes; (3) Ca2+ imaging of the cultured microglia showing predominantly a lack of response to ALS IgG, whereas ATP application elicited a Ca2+ change. This paper also emphasizes possible caveats and cautions regarding critical cell density and purity of cultures, choosing the correct concentration of the Ca2+ dye and dye-loading techniques.

We present here a protocol on how to prepare primary cultures of glial cells, astrocytes, and microglia from rat cortices for time-lapse video imaging of intracellular Ca2+ for research on pathophysiology of amyotrophic lateral sclerosis in the hSOD1G93A rat model.

This protocol demonstrates how to prepare primary cultures of glial cells, astrocytes, and microglia from the cortices of Sprague Dawley rats and how to ...

Coherent Anti-Stokes Raman Spectroscopy (CARS) Application for Imaging Myelination in Brain Slices

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature vibrations of molecules. However, these vibrational signatures are also characteristic of molecules within anatomical tissue such as the brain, making it increasingly useful and applicable for Neuroscience applications. For example, CARS can measure lipids by specifically exciting chemical bonds within these molecules, allowing for quantification of different aspects of tissue, such as myelin involved in neurotransmission. In addition, compared to other techniques typically used to quantify myelin, CARS can also be set up to be compatible with immunofluorescent techniques, allowing for co-labeling with other markers such as sodium channels or other components of synaptic transmission. Myelination changes are an inherently important mechanism in demyelinating diseases such as multiple sclerosis or other neurological conditions such as Fragile X Syndrome or autism spectrum disorders is an emerging area of research. In conclusion, CARS can be utilized in innovative ways to answer pressing questions in Neuroscience and provide evidence for underlying mechanisms related to many different neurological conditions.

Coherent Anti-Stokes Raman Spectroscopy CARS Application for Imaging Myelination in Brain Slices

Visualizing myelination is an important goal for many researchers studying the nervous system. CARS is a technique that is compatible with immunofluorescence that can natively image lipids within tissue such as the brain illuminating specialized structures such as myelin.

Coherent anti-Stokes Raman spectroscopy (CARS) is a technique classically employed by chemists and physicists to produce a coherent signal of signature ...