Name: correlative light and electron microscopy to study microglial interactions with &#946;-amyloid plaques
Uploaded: 2016-06-01T15:00:00
Description: Watch this Scientific Journal Video about Correlative Light and Electron Microscopy to Study Microglial Interactions with Î²-Amyloid Plaques at JoVE.com

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 s...

<ol>
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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...

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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 ...

The overall goal of this procedure is to identify beta-amyloid plaques in brain sections from Alzheimer's disease mouse models prior to pre-embedding immunostaining and tissue processing for electron microscopy. This method can help answer the key questions in the field of neuroimmunology such as the interaction of microglial cell with the synapsis near amyloid beta plots. The main advantage of this technique is that it allows to prescreen brain tissue sections containing amyloid beta plots in the regions and layers of interest.Though this method can be applied to Alzheimer's disease it can also be applied to any other diseases or tissues where amyloidosis is present. First, prepare a five milligrams per milliliter solution of methoxy X04 compound. Using a mirco balance, weight five milligrams of the methoxy X04.Under a fume hood, dissolve the methoxy X04 in 100 microliters of DMSO and stir until a clear, greenish solution is obtained. Successively add 450 microliters of propylene glycol and 450 microliters of phosphate buffered saline while stirring. Keep the solution mixing at four degrees Celsius overnight.The next day, a yellowish-green emulsion should be seen. After injecting methoxy X04, add a dose of 10 milligrams per kilogram of body weight and sacrificing the animal at the desired time point according to the approved methods, wash the para ormaldehyde fixed brain three times with chilled PBS. Then, using a sharp razor blade, remove the olfactory bulb and cut the brain coronally into two to three pieces of approximately equal height, all of which can be sectioned simultaneously in order to accelerate the procedure.Glue the pieces of brain tissue onto the specimen plate secured into the tray. Make sure that the smooth cut surface sticks firmly to the specimen plate. Add PBS solution to the tray until the entire brain surface is completely submerged.Place the tray in the vibratome and adjust the settings of the vibratome to yield 50 micron thick sections. Begin cutting, and if screening immediately transfer the sections into PBS using a fine paintbrush. Alternatively, sections can be placed in glass vials containing cryoprotected solution for storage at 20 degrees Celsius.With the aid of a stereotaxic mouse brain atlas, select brain sections containing the region of interest and place each section into cryoprotectant in a well of a 24-well culture plate. Next, use a disposable pipette to add a droplet of PBS onto a microscope slide and then place the section on the droplet. Examine the section under a fluorescent microscope to identify regions containing methoxy X04 labeled a-beta plaques.Without moving the microscope stage, capture images of the regions of interest in bright field as well as in fluorescence mode. Each image must show the same region in both fields to correlate the plaque to the structural region of the tissue section. Save and name the images taken according to the animal number.Also record the well number in the plate and the field of the pictures taken. Place the section back into the designated well once the imaging is completed. Examine the next section in the sequence using the same procedure to avoid drying the sections.To align the images, open the bright field and UV field images for the same ROI in ImageJ. Then, use the MosaicJ plugin to align the edges of the two images. Save the aligned and combined image according to the well number in a folder with the number of the particular animal.Combine the images from different sections of the same animal to identify and localize the plaques in the region of interest. After the screening process is completed store the examined sections at 20 degrees Celsius is a 24-well culture plate containing cryoprotectant until immunostaining or further processing is carried out. First, prepare 1%osmium tetroxide solution in phosphate buffer in a glass vial.As osmium tetroxide is photosensitive, cover the glass vial with aluminum foil to protect the solution from light. After washing in phosphate buffer, remove the buffer from the sections and spread them flat using a fine paintbrush. 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.Use a transfer pipette to add osmium tetroxide to the sections one drop at a time until the section is immersed. Cover the well with aluminum foil to protect the sections from light and incubate for 30 minutes at room temperature. During this incubation prepare plastic resin in a disposable beaker.Combine the components together in order and mix well using a 10 milliliter serological pipette until a uniform color is obtained. Transfer the prepared mixture to aluminum weighing dishes. These will receive the tissue sections once they have been dehydrated.Once the incubation time has elapsed, dehydrate the sections in increasing concentrations of ethanol for two minutes each. Transfer the dehydrated sections from the 24-well culture plate into 20-milliliter glass vials. Then immerse the sections in propylene oxide three times for two minutes each time to remove residual ethanol.Next, use a bent glass pipette tip or a fine paintbrush to transfer the sections from propylene oxide solution into the plastic resin and leave overnight for infiltration at room temperature. After infiltration, place the aluminum weighing dishes containing the specimens into a 50 to 60 degrees Celsius oven for 10 to 15 minutes. To embed sections on polychlorotrifluoroethylene film sheets first use a fine paintbrush to paint a thin layer of resin onto the section.Then, move one section of tissue at a time from an aluminum weighing dish to the PCTFE film sheet. Remove excess resin from around the tissue, being careful not to disturb it. 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 the two sheets.Polymerize the resin in an incubator for three days at 55 to 60 degrees Celsius. The tissue is then ready for ultrathin sectioning and ultrastructural examination. The following images show dual imaging of one hippocampal section using bright field and fluorescence modes.The regions and layers of interest are visualized under bright field. While the a-beta plaques are successfully localized using a UV filter at a range of 340 to 380 nanometers. This image shows a hippocampal section before pre-embedding immunostaining for IBA1.A visible plaque is outlined by the dotted line. This image shows the same section after pre-embedding immunostaining for IBA1 and processing for transmission electron microscopy. Note that the plaque encircled by a dotted line is still visible upon tissue processing for electron microscopy.Once mastered this protocol can be completed in one week if carried out properly. While attempting this procedure it is important to remember that every step must be done rigorously to reduce the probability of contamination and other structural degredation which will affect the quality of samples. Don't forget that working with osmium can be extremely hazardous and so you should always take precautions such as wearing a lab coat and gloves while performing this procedure.Be sure to discard after using at the end of your experiment in accordance with your facility's guideline.

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