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><tbody><tr><td>Methoxy-X04</td><td>Tocris Bioscience</td><td>4920</td><td>10 mg substrate per tablet</td></tr><tr><td>Propylene glycol</td><td>Sigma Aldrich</td><td>W294004&amp;nbsp;</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher BioReagents</td><td>BP231-1</td><td>Caution: toxic</td></tr><tr><td>Sodium Chloride NaCl</td><td>Sigma</td><td>S9625</td><td></td></tr><tr><td>Sodium phosphate monobasic monohydrate</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Sodium phosphate dibasic</td><td>Sigma</td><td>S0876</td><td></td></tr><tr><td>Tris Hydrochloride</td><td>Fisher BioReagents</td><td>BP153500</td><td></td></tr><tr><td>Acrolein</td><td>Sigma</td><td>110221</td><td>Caution: Toxic</td></tr><tr><td>Paraformaldehyde Granular</td><td>Electron Microscopy Sciences</td><td>19210</td><td>Caution: Toxic</td></tr><tr><td>Filter paper</td><td>Fisher</td><td>09-790-14F</td><td></td></tr><tr><td>Peristaltic Pump with Tubing</td><td>ColeParmer</td><td>cp.78023-00</td><td></td></tr><tr><td>Excel Winged blood Collection Set Needles 25&amp;nbsp;G</td><td>Becton Dickinson</td><td>367341</td><td></td></tr><tr><td>Extrafine Forceps</td><td>F.S.T</td><td>11152-10</td><td>Tip shape: curved</td></tr><tr><td>Scissors</td><td>F.S.T</td><td>14090-09</td><td>Tip shape: straight</td></tr><tr><td>Hartman Hemostats</td><td>F.S.T</td><td>13003-10</td><td>Tip shape: curved</td></tr><tr><td>Surgical Scissors</td><td>F.S.T</td><td>14004-16</td><td>Tip shape: straight</td></tr><tr><td>Micro Dissecting Scissors</td><td>ROBOZ surgical store</td><td>5818</td><td></td></tr><tr><td>Glass scintillation vials</td><td>Fisher Scientific</td><td>74515-20</td><td></td></tr><tr><td>Vibrating Blade Microtome Leica VT1000 S</td><td>Leica Biosystems&amp;nbsp;</td><td>14047235612</td><td></td></tr><tr><td>Vibratome blades</td><td>Electron microscopy Sciences</td><td>71990</td><td></td></tr><tr><td>Microscope Slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr><tr><td>24-well Tissue Culture Plates</td><td>Fisher Scientific</td><td>353047</td><td></td></tr><tr><td>Ethylene Glycol</td><td>Fisher BioReagents</td><td>BP230-4</td><td></td></tr><tr><td>Glycerol</td><td>Fisher BioReagents</td><td>BP229-4</td><td></td></tr><tr><td>Hydrogen Peroxide, 30%</td><td>J.T.BAKER</td><td>cat: 2186-01</td><td></td></tr><tr><td>Sodium borohydride</td><td>Sigma</td><td>480886</td><td></td></tr><tr><td>Tris HCl</td><td>Fisher</td><td>BP153-500ML</td><td></td></tr><tr><td>Fetal bovine Serum (FBS)</td><td>Sigma Aldrich</td><td>F1051</td><td></td></tr><tr><td>Bovine serum albumin (BSA), fraction V</td><td>Thomas Scientific</td><td>C001H24</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Anti IBA1, Rabbit&amp;nbsp;</td><td>WAKO</td><td>019-19741</td><td></td></tr><tr><td>Goat Anti-Rabbit IgG</td><td>Jackson Immunoresearch</td><td>111066046</td><td></td></tr><tr><td>VECTASTAIN Elite ABC Kit (Standard)</td><td>Vector Labs</td><td>PK-6100</td><td></td></tr><tr><td>3.3&#039;-Diaminobenzidine tetra-hydrochloride (DAB)</td><td>Sigma</td><td>D5905-50TAB</td><td>Caution: toxic</td></tr><tr><td>Osmium tetroxide, 4% solution</td><td>Electron Microscopy Sciences</td><td>19150</td><td>Caution: toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component A</td><td>Sigma</td><td>44611</td><td>Resin Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component B</td><td>Sigma</td><td>44612</td><td>Hardener Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component C</td><td>Sigma</td><td>44613</td><td>Plasticizer Caution: Toxic</td></tr><tr><td>Durcupan&amp;trade; ACM single component D</td><td>Sigma</td><td>44614</td><td>Accelerator Caution: Toxic</td></tr><tr><td>Ethanol</td><td>LesAlcoolsdeComerce</td><td>151-01-15N</td><td></td></tr><tr><td>Propylene oxide</td><td>Sigma</td><td>110205</td><td>Caution: corrosive</td></tr><tr><td>Aluminum weigh dishes</td><td>Electron Microscopy Sciences</td><td>70048-01</td><td></td></tr><tr><td>ACLAR&amp;reg;&amp;ndash;Fluoropolymer Films</td><td>Electron Microscopy Sciences</td><td>50425</td><td></td></tr><tr><td>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 Î²-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

11.3K Views.  CHU de Québec Research Center.  Dit artikel beschrijft een protocol voor het visualiseren Ap amyloïde plaques in de ziekte van Alzheimer muismodellen behulp methoxy-X04, die de bloed-hersenbarrière doorkruist en selectief bindt aan P-geplooide platen in dichte kern Ap-plaques. Hiermee pre-screening van plaque bevattende weefselcoupes voorafgaand aan immunokleuring en verwerking voor elektronenmicroscopie. 

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

Imaging Amyloid Tissues Stained with Luminescent Conjugated Oligothiophenes by Hyperspectral Confocal Microscopy and Fluorescence Lifetime Imaging

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find neurodegenerative diseases such as Alzheimer&#39;s and Parkinson&#39;s disease afflicting primarily the central nervous system, and systemic amyloidosis where serum amyloid A, transthyretin and IgG light chains deposit as amyloid in liver, carpal tunnel, spleen, kidney, heart, and other peripheral tissues. Amyloid has been known and studied for more than a century, often using amyloid specific dyes such as Congo red and Thioflavin T (ThT) or Thioflavin (ThS). In this paper, we present heptamer-formyl thiophene acetic acid (hFTAA) as an example of recently developed complements to these dyes called luminescent conjugated oligothiophenes (LCOs). hFTAA is easy to use and is compatible with co-staining in immunofluorescence or with other cellular markers. Extensive research has proven that hFTAA detects a wider range of disease associated protein aggregates than conventional amyloid dyes. In addition, hFTAA can also be applied for optical assignment of distinct aggregated morphotypes to allow studies of amyloid fibril polymorphism. While the imaging methodology applied is optional, we here demonstrate hyperspectral imaging (HIS), laser scanning confocal microscopy and fluorescence lifetime imaging (FLIM). These examples show some of the imaging techniques where LCOs can be used as tools to gain more detailed knowledge of the formation and structural properties of amyloids. An important limitation to the technique is, as for all conventional optical microscopy techniques, the requirement for microscopic size of aggregates to allow detection. Furthermore, the aggregate should comprise a repetitive &#946;-sheet structure to allow for hFTAA binding. Excessive fixation and/or epitope exposure that modify the aggregate structure or conformation can render poor hFTAA binding and hence pose limitations to accurate imaging.

Amyloid deposition is a hallmark of different diseases and afflicts many different organs. This paper describes the application of luminescent conjugated oligothiophene fluorescence staining in combination with fluorescence microscopy techniques. This staining method represents a powerful tool for detection and exploration of protein aggregates in both clinical and scientific setups.

Proteins that deposit as amyloid in tissues throughout the body can be the cause or consequence of a large number of diseases. Among these we find ...

Biochemistry

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Bioengineering

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of epoxy embedded human pathology tissue. We employ commercial antibody fragment conjugated QDs that are visualized by widefield fluorescence light microscopy and transmission electron microscopy.

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using ...

Quantitative 3D In Silico Modeling (q3DISM) of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible for phagocytosis and clearance of debris and detritus. These cells include CNS-resident macrophages known as microglia, and mononuclear phagocytes infiltrating from the periphery. Light microscopy has generally been used to visualize phagocytosis in rodent or human brain specimens. However, qualitative methods have not provided definitive evidence of in vivo phagocytosis. Here, we describe quantitative 3D in silico modeling (q3DISM), a robust method allowing for true 3D quantitation of amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent Alzheimer&#39;s Disease (AD) models. The method involves fluorescently visualizing A&#946; encapsulated within phagolysosomes in rodent brain sections. Large z-dimensional confocal datasets are then 3D reconstructed for quantitation of A&#946; spatially colocalized within the phagolysosome. We demonstrate the successful application of q3DISM to mouse and rat brains, but this methodology can be extended to virtually any phagocytic event in any tissue.

Quantitative 3D In Silico Modeling q3DISM of Cerebral Amyloid-beta Phagocytosis in Rodent Models of Alzheimer's Disease

We developed a methodology for quantitative 3D in silico modeling (q3DISM) of cerebral amyloid-&#946; (A&#946;) phagocytosis by mononuclear phagocytes in rodent models of Alzheimer&#39;s disease. This method can be generalized for the quantitation of virtually any phagocytic event in vivo.

Neuroinflammation is now recognized as a major etiological factor in neurodegenerative disease. Mononuclear phagocytes are innate immune cells responsible ...

Medicine

Visualizing Axonal Growth Cone Collapse and Early Amyloid &#946; Effects in Cultured Mouse Neurons

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the brains of AD patients, these do not improve memory functions. Since A&#946; aggregates in the brain before the appearance of memory impairments, targeting A&#946; may be inefficient for treating AD patients who already exhibit memory deficits. Therefore, downstream signaling due to A&#946; deposition should be blocked before AD development. A&#946; induces axonal degeneration, leading to the disruption of neuronal networks and memory impairments. Although there are many studies on the mechanisms of A&#946; toxicity, the source of A&#946; toxicity remains unknown. To help identify the source, we propose a novel protocol that uses microscopy, gene transfection, and live cell imaging to investigate early changes caused by A&#946; in axonal growth cones of cultured neurons. This protocol revealed that A&#946; induced clathrin-mediated endocytosis in axonal growth cones followed by growth cone collapse, demonstrating that inhibition of endocytosis prevents A&#946; toxicity. This protocol will be useful in studying the early effects of A&#946; and may lead to more efficient and preventative AD treatment.

Here a protocol to investigate the early effects of amyloid-&#946; (A&#946;) in the brain is presented. This shows that A&#946; induces clathrin-mediated endocytosis and collapse of axonal growth cones. The protocol is useful in studying early effects of A&#946; on axonal growth cones and may facilitate prevention of Alzheimer's disease.

Amyloid-&#946; (A&#946;) causes memory impairments in Alzheimer's disease (AD). Although therapeutics have been shown to reduce A&#946; levels in the ...

Immunofluorescence Staining Using IBA1 and TMEM119 for Microglial Density, Morphology and Peripheral Myeloid Cell Infiltration Analysis in Mouse Brain

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia selectively), when combined with IBA1 (which provides an exceptional visualization of their morphology), allows investigation of changes in density, distribution, and morphology. Quantifying these parameters is important in providing insights into the roles exerted by microglia, the resident macrophages of the brain. Under normal physiological conditions, microglia are regularly distributed in a mosaic-like pattern and present a small soma with ramified processes. Nevertheless, as a response to environmental factors (i.e., trauma, infection, disease, or injury), microglial density, distribution, and morphology are altered in various manners, depending on the insult. Additionally, the described double-staining method allows visualization of infiltrating macrophages in the brain based on their expression of IBA1 and without colocalization with TMEM119. This approach thus allows discrimination between microglia and infiltrating macrophages, which is required to provide functional insights into their distinct involvement in brain homeostasis across various contexts of health and disease. This protocol integrates the latest findings in neuroimmunology that pertain to the identification of selective markers. It also serves as a useful tool for both experienced neuroimmunologists and researchers seeking to integrate neuroimmunology into projects.

This protocol describes a step-by-step workflow for immunofluorescent costaining of IBA1 and TMEM119, in addition to analysis of microglial density, distribution, and morphology, as well as peripheral myeloid cell infiltration in mouse brain tissue.

This is a protocol for the dual visualization of microglia and infiltrating macrophages in mouse brain tissue. TMEM119 (which labels microglia ...

Immunology and Infection

Detecting Amyloid-&#946; Accumulation via Immunofluorescent Staining in a Mouse Model of Alzheimer's Disease

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies on accumulation of amyloid-&#946; (A&#946;) in the brain. A&#946; is produced from the proteolytic cleavage of the beta-amyloid precursor protein (APP) by &#946;-secretase and &#947;-secretase. In pathological circumstances, the increased &#946;-cleavage of APP leads to overproduction of A&#946;, which aggregates into A&#946; plaques. Since A&#946; plaques are a characteristic of AD pathology, detecting the amount of A&#946; is very important in AD research. In this protocol, we introduce the immunofluorescent staining method to visualize A&#946; deposition. The mouse model used in our experiments is 5&#215;FAD, which carries five mutations found in human familial AD. The neuropathological and behavioral deficits of 5xFAD mice are well-documented, which makes it a good animal model to study A&#946; pathology. We will introduce the procedure including transcardial perfusion, cryosectioning, immunofluorescent staining and quantification to detect A&#946; accumulation in 5&#215;FAD mice. With this protocol, researchers can investigate A&#946; pathology in an AD mouse model.

In the neuropathology of Alzheimer's disease, one of the most crucial characteristics is the deposition of amyloid-&#946;. In this protocol, we describe the method of immunofluorescent staining in 5&#215;FAD transgenic mouse to detect amyloid-&#946; accumulation in plaques. The process of perfusion, cryosectioning, staining and quantification will be described in detail.

Alzheimer's disease (AD) is a neurodegenerative disease that contributes to 60-70% dementia around the world. One of the hallmarks of AD undoubtedly lies ...

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. Under homeostatic conditions, microglia possess a highly ramified morphology. This facilitates their monitoring of the surrounding environment through the continuous extending and retracting of their processes. During brain injury and inflammation, however, microglia become activated and undergo dramatic morphological changes, retracting their ramified processes and swelling their cell body. This facilitates activities such as migration and phagocytosis, which microglia undertake to navigate the brain environment to a less pathological state.
This close relationship between microglial morphology and changes in their activity have enabled considerable insights into various microglial functions. However, such morphological and activity changes are themselves phenomena that can result from any number of intracellular signaling pathways. Moreover, the time-lag between stimulus and response, as well as the highly compartmentalized morphology of microglia, make it difficult to isolate the causative mechanisms that underpin function. To solve this problem, we developed a genetically modified mouse line in which a highly sensitive fluorescent Ca2+-indicator protein is specifically expressed in microglia.
After describing methods for in vivo microglial Ca2+ imaging, this paper presents a structured analysis approach that classifies this Ca2+ activity to rationally defined subcellular regions, thus ensuring that the spatial and temporal dimensions of the encoded information are meaningfully extracted. We believe that this approach will provide a detailed understanding of the intracellular signaling rules that govern the diverse array of microglial activities associated with both higher brain functions and pathological conditions.

Spatiotemporal Analysis of Microglial Ca2+ Activity at Single-Cell Resolution

In this paper, we describe a protocol for in vivo imaging of microglial Ca2+ activity and subsequent analysis of its spatiotemporal dynamics. This method enables thorough characterization of how microglia respond to changes in the brain environment, appropriately capturing the fine spatiotemporal scales at which such events occur.

Microglia are the sole resident immune cells in the central nervous system. Their morphology is highly plastic, changing depending on their activity. ...

Two-Photon Microscopy for Studying Microglial Process Attraction Toward a Compound in a Mouse Brain Slice

<a href='https://app.jove.com/v/58788/two-photon-imaging-microglial-processes-attraction-toward-atp-or'>Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. (2019)</a>This video demonstrates the use of two-photon microscopy to study the attraction of microglial processes toward an injected compound in brain slices. It provides an overview of preparing mouse brain slices, injecting a chemoattractant test compound, and imaging fluorescently labeled microglia to visualize their responses.

Source: Etienne, F. et al. Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices.&#160;J. Vis. Exp. ...

JoVE Encyclopedia of Experiments

Neuroimaging

Live Imaging & In Vivo Tracking

Visualization and Mapping of Methoxy-X04-Labeled Amyloid Plaques in an Alzheimer’s Disease Mouse Brain Section

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/54060/correlative-light-and-electron-microscopy-to-study-microglial-interactions-with-946-amyloid-plaques'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Bisht, K.,&#160;et. al<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>. Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'> (2016)</a> &#160;This video demonstrates the visualization and mapping of Methoxy-X04-labeled amyloid plaques in Alzheimer&#8217;s disease mouse brain sections using fluorescence and bright-field microscopy, enabling precise correlation of plaques with anatomical structures for studying Alzheimer&#8217;s pathology.

Source:&#160;Bisht, K.,&#160;et. al. Correlative Light and Electron Microscopy to Study Microglial Interactions with &#946;-Amyloid Plaques.&#160;J. Vis. ...

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

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