This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, 401848-2011 to M.P.). M.P received a career award from the Fonds de recherche du Qu&#233;bec-Sant&#233; (FRQ-S). L.E. was the recipient of a doctoral fellowship from the FRQ-S (FRQ-S 14D 29441). We thank Marie-Jos&#233;e Wallman for technical assistance.

Ethics Statement: All protocols involving animals were approved by the Comit&#233; de Protection des Animaux de l&#39;Universit&#233; Laval and were made in accordance with the Canadian Council on Animal Care&#39;s Guide to the Care and Use of Experimental Animals (Ed. 2). The protocol described here was optimized for adult animals of approximately 800 g. The volumes of fixative should be adjusted according to the animal&#39;s size.
1. Preparation of Solutions for Transcardiac Perfusion
<ol>
	<li>Prepare 1 L of a 50 mM sodium Phosphate-buffered Saline (PBS) solution according to the following steps. Prepare the solution at most 24 h before perfusion and keep at 4 &#176;C until use.
	<ol>
		<li>Measure 800 mL of distilled water in a 1 L beaker. Add 5.87 g of dibasic anhydrous sodium phosphate (Na2HPO4), 1.20 g of monobasic monohydrate sodium phosphate (NaH2PO4&#183;H2O), and 9 g of sodium chloride (NaCl). Stir to dissolve.</li>
		<li>Adjust the pH to 7.4 by gradually adding 5 N NaOH. Add distilled water to reach a total volume of 1 L. 
		CAUTION: NaOH is a corrosive chemical compound. Wear the appropriate Personal Protective Equipment (PPE; laboratory coat, gloves, protective goggles, etc.).</li>
	</ol>
	</li>
	<li>Prepare 2 L of 4% paraformaldehyde (PFA) according to the following steps. This solution should be prepared at most 24 h before surgery and kept at 4 &#176;C until use.
	<ol>
		<li>Measure 1.5 L of distilled water in a 2-L beaker. Add 23.48 g of dibasic anhydrous sodium phosphate (Na2HPO4) and 4.80 g of monobasic monohydrate sodium phosphate (NaH2PO4&#183;H2O). Stir to dissolve. Add distilled water to reach a total volume of 2 L.</li>
		<li>Heat the solution under a venting hood until the temperature reaches approximately 45 - 55 &#176;C. Do not heat to over 60 &#176;C.</li>
		<li>Gradually add 80 g of PFA to the solution and stir until completely dissolved (approximately 30 - 60 min). Keep monitoring the temperature to keep it below 60 &#176;C. 
		CAUTION: PFA is highly volatile in its powder form. It is highly toxic if in contact with eyes or skin and is hazardous in case of inhalation or ingestion. Wear PPE and use with caution under a venting hood.</li>
		<li>Cool the solution down to 4 &#176;C and filter. Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Prepare 1 L of 3% acrolein in 0.1 M Phosphate Buffer (PB) according to the following steps. The PB solution should be prepared at most 24 h prior to perfusion. 
	CAUTION: Acrolein is highly toxic if inhaled and can produce immediate damage. It is also corrosive and highly toxic if absorbed through the skin. It is carcinogen and mutagenic. Wear the appropriate PPE and use under a venting&#160;hood.
	<ol>
		<li>Measure 800 mL of distilled water. Add 8.66 g of dibasic anhydrous sodium phosphate (Na2HPO4) and 5.38 g of monobasic monohydrate sodium phosphate (NaH2PO4&#183;H2O). Stir until all the salts dissolve. Add distilled water to reach a total volume of 1 L. Keep the solution at 4 &#176;C.</li>
		<li>Before surgery, transfer 900 mL of the PB solution to a 1 L glass container and add 30.94 mL of 97% acrolein solution under a venting hood. Add PB solution to reach a total volume of 1 L and stir. Filter the solution and keep it at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Transcardiac Perfusion and Brain Dissection
<ol>
	<li>Keep solutions on ice during the entire surgical procedure. Prepare the pump by placing the tube in the first solution to be used (PBS; 50 mM) and turning the pump on until no air remains in the hose. For the transcardiac perfusion of a macaque monkey, use a 21 G needle and set the outflow at approximately 80 mL/min.</li>
	<li>Anesthetize the animal with an intramuscular injection of a mixture of ketamine (20 mg/kg), xylazine (4 mg/kg), and acepromazine (0.5 mg/kg). Maintain the animal under isoflurane (3%) sedation.</li>
	<li>Attach the animal&#39;s limbs to a venting table.</li>
	<li>With a scalpel, remove the skin up to the armpits. Cut the abdominal muscles. Use heavy-duty surgical scissors to cut the ribs laterally by carefully avoiding the vital organs.</li>
	<li>Cut the diaphragm with surgical scissors and raise the rib cage to expose the heart. Once the diaphragm is cut, proceed quickly, since the heart will stop beating within minutes.</li>
	<li>Remove the pericardium with a scalpel blade and insert the needle into the left ventricle. With a scalpel, carefully make a small excision to the right atrium. Quickly start the pump at 72 mL/min and hold the needle in place. If possible, tilt the animal in order to have its head on a lower level than the heart. 
	NOTE: Be careful not to pierce the interventricular septum when inserting the needle.
	<ol>
		<li>Rinse the blood with approximately 300 mL of PBS until the lungs are white and no blood comes out of the right atrium.</li>
	</ol>
	</li>
	<li>Stop the pump, quickly transfer the hose/tube to the 3% acrolein solution, and start the pump again. Gradually increase the pumping speed to 80 mL/min once the heart has stopped beating. Perfuse approximately 500 mL of the acrolein solution.</li>
	<li>Stop the pump, quickly transfer the hose to the 4% PFA solution, and start the pump again. This step requires approximately 1 L of PFA. 
	NOTE: The fixation is complete when the forelimbs are rigid and the neck is stiff.</li>
	<li>Cut the head and carefully dissect the brain out of the skull. Take care to not damage the brain with the surgical instruments. The perfusion is optimal when the brain is pale (no trace of blood) and rigid (Figure 1A).</li>
	<li>Immerse the intact brain in 4% PFA for 1 h at 4 &#176;C.</li>
	<li>Serially cut the brain with a cooling vibratome (4 &#176;C) in the desired plane into 50 &#181;m thick sections and collect them in PBS (0.1 M) (Figure 1B). 
	NOTE: This step can be carried out in many ways according to the desired protocol. Hemispheres can be separated prior to vibratome cutting, or kept whole. If the brain is too large for the vibratome platform, it can be cut into smaller blocks. After this step, sections can be stored for a long period of time at -30 &#176;C in an antifreeze solution made of 40% PB (50 mM), 30% glycerol, and 30% ethylene glycol.</li>
</ol>
3. Pre-embedding Immunohistochemistry (Figure 1C)
<ol>
	<li>Prepare a 4 L stock solution of Tris-buffered Saline (TBS, 50 mM, pH 7.6) as follows.
	<ol>
		<li>Measure 2L of distilled water in a 4 L beaker, add 24.23 g of trihydroxymethyl aminomethane (THAM; C4H11NO3), and stir to dissolve.</li>
		<li>Adjust the pH to 7.6 with approximately 148 mL of 1 N HCl. The acid should be added with caution to avoid reaching a pH below 7.6. The total volume should be 4 L. 
		CAUTION: HCl is highly corrosive. Wear the appropriate PPE.</li>
	</ol>
	</li>
	<li>Select sections (from step 2.11) containing the region of interest to be processed for EM immunohistochemistry.</li>
	<li>Wash the free-floating sections 3x&#160;in PBS (0.1 M, pH 7.4) for 5 min at RT&#160;to rinse the antifreeze solution.</li>
	<li>Prepare a 0.5% solution of NaBH4 diluted in PBS.
	<ol>
		<li>Weigh 0.05 g of NaBH4 and dilute it in 10 mL of PBS. Do not cover. Prepare this solution just before use.</li>
	</ol>
	</li>
	<li>Incubate the sections in the freshly prepared NaBH4 solution for 30 min at RT. Rock gently. Do not cover.</li>
	<li>Wash 3x&#160;in PBS for 10 min at RT, rocking vigorously until none of the reaction gas remains.</li>
	<li>Prepare a blocking solution for EM with 2% appropriate normal serum and 0.5% cold fish gelatin diluted in PBS. 
	NOTE: The quantity should be calculated in order to have enough for the following three steps. Use a serum made from the same animal species hosting the secondary antibody.&#160;Avoid the use of antigen retrieval methods or addition of small amounts of triton (to increase the penetration of antibodies), as these methods significantly compromise the quality of the tissue.</li>
	<li>Incubate the sections in the blocking solution for 1 h at RT. Rock gently.</li>
	<li>Prepare primary antibody solution diluted in the blocking solution. 
	NOTE: The concentration of the primary antibody is usually the same as for LM immunohistochemistry, but consider conducting tests with different antibody concentrations beforehand, as some primary antibodies may not work with acrolein. If so, it is possible to use a mix of glutaraldehyde (0.1 - 2%) and 4% PFA for transcardiac perfusion and obtain similar results. Optimize the incubation time and temperature as well.</li>
	<li>Incubate the sections in primary antibody solution overnight at RT with gentle rocking. Use a previously tested incubation time and temperature.</li>
	<li>Wash the sections 3x in PBS for 5 min at RT with gentle rocking.</li>
	<li>Prepare a 1:1,000 solution of biotinylated secondary antibody diluted in the blocking solution. 
	NOTE: The secondary antibody must be raised against the host species used to generate the primary antibody.</li>
	<li>Incubate the sections in secondary antibody solution for 1.5 h at RT. Rock gently.</li>
	<li>Prepare an avidin-biotin-peroxidase (ABC) solution at least 60 min prior to the end of the secondary antibody incubation.
	<ol>
		<li>Use a calibrated pipette to measure 8.80 &#181;L/mL of solutions A and B and dilute them in PBS.</li>
		<li>Rock mildly for at least 60 min at RT to allow the complete binding between the avidin and biotin molecules.</li>
	</ol>
	</li>
	<li>After the incubation in secondary antibody solution, wash 3x in PBS for 10 min at RT.</li>
	<li>Incubate the sections in the ABC solution for 1 h at RT. Rock gently.</li>
	<li>Wash once in PBS and twice in TBS for 10 min at RT with gentle rocking.</li>
	<li>Prepare a fresh solution of 0.05% 3,3&#39;-diaminobenzidine (DAB) with 0.005% H2O2 diluted in TBS.
	<ol>
		<li>Weigh 12.5 mg of DAB and dilute it in 25 mL of cold TBS. Protect from light. 
		CAUTION: The DAB powder is highly volatile and harmful if inhaled. It is carcinogenic and teratogenic. Thus, pregnant or nursing women should not manipulate this product, even when diluted. Use a N95 mask when manipulating and wear PPE.</li>
		<li>Filter the solution and add 4.5 &#181;L of 30% H2O2 just before use.</li>
	</ol>
	</li>
	<li>Incubate sections in the DAB solution for 3 to 7 min at RT. Rock gently. 
	NOTE: The brown precipitate should not be too dark in order to avoid a high level of background staining. The incubation time should be optimized accordingly.</li>
	<li>Stop the reaction by quickly washing twice in cold TBS, then twice for 10 min in cold TBS at RT, followed by twice for 10 min in PB, with mild rocking. 
	NOTE: The use of PB (and not PBS) is critical in order to eliminate any traces of NaCl, as it would react with osmium and form crystals.</li>
</ol>
4. Osmification and Embedding for Electron Microscopic Observation
<ol>
	<li>Prepare a solution of 1% osmium tetroxide (OsO4) diluted in PB. Protect from light. 
	CAUTION: Osmium is highly toxic and should not come in contact with the skin, eyes, or mouth and should not be inhaled. It can cause death if ingested. It should only be used under a venting hood and with appropriate PPE.</li>
	<li>Incubate the sections in OsO4 solution for 30 min at RT under the venting hood (without agitation) and cover them with aluminum foil to protect them from light. Completely flatten the sections prior to adding the OsO4 solution. 
	NOTE: The sections become very dark and rigid and should be manipulated with care following this step.</li>
	<li>Prepare water-repellent epoxy resin during the osmification.
	<ol>
		<li>Add appropriate amounts of each component of the epoxy resin mix (20 g of epoxy resin, 20 g of hardener, 0.6 g of accelerator and 0.4 g of plasticizer) to a large plastic cup. Stir with a wooden stick or plastic pipette until a homogeneous brown color is obtained. 
		NOTE: It is critical to use the exact proportion of each component.</li>
		<li>Transfer equal quantities to aluminum cups of appropriate sizes, depending on the number of sections to be processed. Allow it to rest.</li>
	</ol>
	</li>
	<li>Wash the osmificated sections 3x in PB for 10 min at RT with low-speed rocking.</li>
	<li>Dehydrate the sections in the following series of graded ethanol for 2 min each: 2 times in 35% ethanol; 1 time each in 50, 70, 80, 90, and 95% ethanol; and 3x&#160;in 100% ethanol.</li>
	<li>Transfer the sections to glass vials to complete the dehydration process by incubating the sections 3 times for 2 min in propylene oxide. 
	CAUTION: Propylene oxide is a highly volatile and toxic organic solvent. It can cause serious damage in the eyes or skin in case of contact or if inhaled or ingested. It has been classified as a grade 2 carcinogenic substance. It should only be used under a venting hood and with PPE. It is also highly flammable and should be kept away from any heat source. 
	NOTE: Prior to this step, sections should be carefully transferred in glass vials, since propylene oxide is an organic solvent and is incompatible with plastic. At this point, sections are very fragile and should be manipulated with care. Sections can be alternatively transferred to glass vials prior to step 4.5.</li>
	<li>Transfer sections carefully, one by one, in the aluminum cups and avoid contact with air as much as possible. Flat-embed the sections in previously mixed water-repellent epoxy resin and incubate them overnight under the venting hood at RT. 
	NOTE: At this step, the sections are completely dehydrated and very fragile and should be manipulated with care.</li>
	<li>Using mineral oil, prepare grease-coated glass slides along with greased plastic coverslips.</li>
	<li>Soften the resin by incubating aluminum cups at 60 &#176;C for 12-15 min at most. Carefully flatten the sections on the greased side of the glass slide. Place the greased coverslip and carefully push out any remaining air.</li>
	<li>Incubate the slides at 60 &#176;C for 48 h. 
	NOTE: It is critical to not exceed 48 h incubation time, as the resin will become too hard.</li>
	<li>Remove the plastic coverslip.</li>
</ol>
5. Sample Preparation for Ultrathin Sectioning and Observation Using a Transmission Electron Microscope
<ol>
	<li>Use binoculars to find the region of interest and cut a small quadrangular piece of approximately 1 mm2 with a scalpel.</li>
	<li>File the tip of a resin block and glue the quadrangular piece onto it (Figure 1C). Allow the glue to dry for at least 1 h or overnight prior to sectioning.</li>
	<li>Using an ultramicrotome, cut the quadrangular piece into 80 &#181;m-thick sections (Figure 1D).
	<ol>
		<li>Place the resin block in an ultramicrotome deck in the vertical position and, using a sharp razor blade, gradually cut each side of the resin block to form a trapezoid with smooth sides.</li>
		<li>Put the deck in its horizontal position and rotate the block until the longest side of the trapezoid is facing downward.</li>
		<li>Use a diamond trimming tool or a glass knife to trim the surface of the quadrangular piece. Set the ultramicrotome to cut 300 &#181;m thick sections at 1 mm/s. Adjust the knife to be vertically parallel to the quadrangular piece and to display a very small horizontal angle of approximately 1&#176;. 
		NOTE: This angle will allow the user to approach the tissue almost parallel to the surface of the block, where immunolabeled elements are more likely to be found. When using these parameters with the diamond trimming tool, the resin displays a shiny white color. When the tissue is being cut, it changes to a purple or greenish color.&#160;</li>
		<li>Use an ultra 45&#176; diamond knife equipped with a boat filled with distilled water to cut 80 &#181;m thick sections,&#160;smoothen sections by passing over them with a piece of absorbing paper tipped in xylene, and collect serial sections on formvar-coated nickel slot grids or bare 150 mesh copper grids (Figure 1E).</li>
	</ol>
	</li>
	<li>Place the grids in a grid storage box.</li>
	<li>Stain the grids with lead citrate.
	<ol>
		<li>Use a 5 mL syringe and a 0.2 &#181;m syringe filter to prepare a 1:1 solution of filtered lead citrate stock solution and filtered distilled water. Protect it from light. 
		NOTE: The stock solution of lead citrate should be made fresh every month to avoid the formation of solid deposits. See the material data sheet for the stock recipe. In addition, if many series of grids need to be stained, change the diluted solution when it becomes milky.</li>
		<li>Place each grid onto a drop of the diluted solution, with the section in contact with the solution. Incubate for 3 min.</li>
		<li>Use small tweezers to hold the grid, and thoroughly rinse it in two beakers containing distilled water.</li>
		<li>Remove excess water by gently using absorbing paper. Store the grids in a grid box. Wait 30 min before examining the sections by Transmission Electron Microscopy (TEM; Figure 1F).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In this article, we present a reliable protocol for transcardiac perfusion of non-human primates and pre-embedding immunohistochemistry suitable for EM sample examination. Although typical cryo-EM, such as CEMOVIS, provides a good preservation of brain ultrastructure, it also limits the use of immunohistochemistry12. Other techniques, including cryo-substitution and Tokuyaso technique, allow post-embedding immunohistochemistry, but these techniques are expensive due to additional devices needed du...

Light microscopy, including confocal and two-photon microscopy, has proven to be an efficient tool for studying in vivo neuronal processes, among other things1,2. Although the typical spatial resolution at the Light Microscopic (LM) level is approximately 200 nm, recent technological advances using different light sources, such as extreme ultraviolet and soft X-ray microscopy, have notably increased this resolution to a nearly 10 nm spatial resolution3,4,5. Other technological advances in imaging include combined magnetic resonance imaging with histology and provide a novel method for measuring the thickness of the myelin sheath in vivo, a parameter that was traditionally measurable only at the Electron Microscopic (EM) level6,7. Although these advances at the LM level provide an excellent tool for studying living processes, a detailed view and characterization of structures, such as synaptic contacts, can only be achieved with EM, which offers a resolution that can reach 0.5 nm. However, observation at the EM level requires the specimens to be dead and altered in some ways, with chemical fixatives and dehydration processes, in order to preserve the cytoarchitecture. Thus, examining biological samples at high resolution can be challenging due to radiation damage from the electron beam, low contrast, structural deviations of membranes, or even the presence of artifacts that can occur following dehydration and epoxy embedding8,9,10.
Preserving specimens in their native form for structural analysis can be achieved by using &#34;Cryo EM of Vitrified Sections&#34; or CEMOVIS, a sectioning approach that involves rapidly freezing and embedding the sample in vitreous ice and examining the sections under the EM at a cryogenic temperature11,12. This procedure allows for the examination of samples while they are still solid and fully hydrated, thus eliminating artifacts caused by dehydration processes13. However, this method involves additional devices for cryo-ultramicrotomy, as well as additional devices on the standard EM, in order to allow this observation at very low temperatures, which generate significant additional costs. In addition, the CEMOVIS approach precludes the use of immunolabeling techniques, since antibodies usually have to be incubated at RT. Alternatively, it is possible to combine ultrastructural analysis with immunohistochemical procedures by using a freeze-substitution approach, during which cryo-fixed specimens are slowly thawed while immerged in cryo-protective chemicals and are then embedded in specialized resins, such as Lowicryls. Post-embedding immunolabeling can then be performed on such material12. However, freeze-substitution and cryo-fixation techniques are time consuming. They require the installation of additional equipment and still require samples to be exposed to organic solvent and chemical fixative that can alter the cytoarchitecture, despite the use of a low temperature14,15. Hence, despite all the technological advances both at the LM and EM level, chemical fixation of brain tissue, particularly with acrolein, remains a low-cost and time-efficient method to combine immunohistochemistry with EM16.
In the last decades, many attempts were made to find a mixture of aldehydes that provide the best tissue preservation. Before the 1960s, the only chemical fixative that gave acceptable results for EM was osmium tetroxide. However, osmium tetroxide is highly toxic and expensive, precluding its use through the vascular system to fix organs such as the brain. Acrolein was introduced in the late 1950s as a reliable method for animal tissue preservation suitable for EM observation of cellular structures17. It penetrates the tissue more deeply and reacts more quickly than other aldehydes when used for fixation by immersion and allows good preservation of cytoplasmic components, with minimal shrinkage of the tissue17. Such a feature gives acrolein fixation a clear advantage over other aldehydes when used in fresh tissue, by allowing a more accurate localization of living molecular compounds, such as enzymes and other proteins18. Indeed, it has been validated through the years as an easy, efficient and low-cost method of fixation for visualization at the EM level in many species, including amphibians and rodents, as it efficiently stabilizes peptides and proteins, retains antigenicity and provides relatively intact ultrastructure when used in combination with another aldehyde fixative16,18,19,20,21. Protocols for acrolein fixation in rodents have since then been standardized and used extensively, particularly by the Pickel group, to implement dual immunolabeling for EM16,22. A few groups have used acrolein fixation in non-human primate brain tissue23. However, to our knowledge, there is only one published protocol efficiently describing chemical fixation with acrolein in non-human primates that is compatible with EM immunolabeling24.
In this article, we provide an easy and reliable method to efficiently chemically fix non-human primate brains with acrolein, allowing for a potentially long-term preservation along with pre-embedding immunolabeling and transmission EM examination.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="46">
			<td height="46" style="height:46px;width:216px;">Dibasic anhydrous sodium phosphate (Na2HPO4)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">S374-500</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:216px;">Monobasic monohydrate sodium phosphate (NaH2PO4.H2O)</td>
			<td style="width:123px;">EM Science</td>
			<td style="width:119px;">SX0710-1</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride (NaCl)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">S271-3</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hydroxymethyl aminomethane (THAM)</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">T370-500</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">HCl&#160;</td>
			<td style="width:123px;">EMD</td>
			<td style="width:119px;">HX0603-3</td>
			<td style="width:177px;">1N dilution. Product is corrosive. Use with appropriate potection.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">NaOH</td>
			<td style="width:123px;">EMD</td>
			<td style="width:119px;">SX0590-1</td>
			<td style="width:177px;">5N dilution. Product is corrosive. Use with appropriate protection.</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Paraformaldehyde</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">P6148</td>
			<td style="width:177px;">4% dilution. Product is highly volatile in its powder form and highly toxic. Use with caution under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Acrolein (90%)</td>
			<td style="width:123px;">sigma</td>
			<td align="right" style="width:119px;">110221</td>
			<td style="width:177px;">3% dilution. Product is highly toxic. Use under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Autopsy venting table</td>
			<td style="width:123px;">Mopec</td>
			<td style="width:119px;">CE400</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Electronic perfusion pump</td>
			<td style="width:123px;">cole parmer</td>
			<td style="width:119px;">masterflex L/S 7523-90</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle (perfusion)</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">NN-1838R</td>
			<td style="width:177px;">18G 1 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Needle</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">NN-2713R</td>
			<td style="width:177px;">21G 1/2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ketamine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">20 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylazine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">4 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acepromazine</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;">0.5 mg/kg</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Scalpel blades</td>
			<td style="width:123px;">Feather&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; lance</td>
			<td style="width:119px;">201011&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; J9913</td>
			<td style="width:177px;">No.22 for surgery and&#160;&#160;&#160; No. 11 for EM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Surgical scissors</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rongeurs</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Vibratome</td>
			<td style="width:123px;">Leica</td>
			<td style="width:119px;">VT 1200S</td>
			<td style="width:177px;">Calibrate blade before each use, when the device allows it</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vibratome razor blade</td>
			<td style="width:123px;">Gillette</td>
			<td style="width:119px;">GIN 642107</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycerol</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">G33-4</td>
			<td style="width:177px;">30% dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethylene glycol</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">E178-4</td>
			<td style="width:177px;">30% dilution</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Sodium borohydride (NaBH4)</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">S-9125</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Normal horse serum</td>
			<td style="width:123px;">Jackson immunoResearch Laboratories</td>
			<td>008-000-121</td>
			<td style="width:177px;">2% dilution</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cold-fish gelatin</td>
			<td style="width:123px;">Aurion</td>
			<td style="width:119px;">900.033</td>
			<td style="width:177px;">0.5% dilution. Original product is concentrated at 40%</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primary antibody, SERT</td>
			<td style="width:123px;">Santa Cruz biotechnology</td>
			<td style="width:119px;">SC-1458</td>
			<td style="width:177px;">1/500 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Primary antibody, ChAT</td>
			<td style="width:123px;">Chemicon (Millipore)</td>
			<td style="width:119px;">AB144P</td>
			<td style="width:177px;">1/25 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Primary antibody, TH</td>
			<td style="width:123px;">ImmunoStar</td>
			<td style="width:119px;">22941</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated secondary antibody, goat</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">BA-9500</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Biotinylated secondary antibody, mouse</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">BA-2000</td>
			<td style="width:177px;">1/1000 dilution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectastain elite ABC kit</td>
			<td style="width:123px;">Vector laboratories</td>
			<td style="width:119px;">PK6100</td>
			<td style="width:177px;">8.8 &#181;L/mL of A and B each</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">3&#39;3 diaminobenzidine (DAB)</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">D5637</td>
			<td style="width:177px;">0,05% dilution. Product is highly volatile in its powder form and toxic. Do not throw waste in the sink.</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">Peroxide (H2O2) 30%</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">H-323</td>
			<td style="width:177px;">0,005% dilution</td>
		</tr>
		<tr height="212">
			<td height="212" style="height:212px;width:216px;">Osmium tetroxide (OsO4)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">2% 19152&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 4% 19150&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;</td>
			<td style="width:177px;">Original solution can be either 2% ou 4%. Keep attention to which one is used to calculate the final 1% dilution. Product is very sensitive to light. Osmium is highly toxic. Use only under a venting hood with appropriate protection.</td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:216px;">Durcupan water-repellent epoxy resin</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">A: M epoxy resin (44611)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; B: hardener 964 (44612)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; C: accelerator 960 (DY 060)&#160; (44613)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; D: plasticizer (44614)&#160;</td>
			<td style="width:177px;">Polymerize 48h at 58 &#176;C before throwing in waste.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Alumium cups</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">70048-01</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ethanol</td>
			<td style="width:123px;">commercial alcohols</td>
			<td style="width:119px;">1019C</td>
			<td style="width:177px;">Dilute in distilled water with appropriate concentration</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Propylene oxide</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">20401</td>
			<td style="width:177px;">Organic solvent. Highly volatile and toxic. Use under a venting hood.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-coated medium glass slides</td>
			<td style="width:123px;">brain research laboratories</td>
			<td style="width:119px;">3875-FR</td>
			<td style="width:177px;">Grease surface with mineral oil</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Plastic film (Aclar embedding film)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">50425-25</td>
			<td style="width:177px;">Grease surface with mineral oil</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ultramicrotome</td>
			<td style="width:123px;">Leica UC7</td>
			<td style="width:119px;">EM UC7</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diamond trimming tool (ultratrim)</td>
			<td style="width:123px;">Diatome&#160;</td>
			<td style="width:119px;">UT 1081</td>
			<td style="width:177px;">Can use glass knife alternatively</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ultra 45&#176; Diatome Diamond knife</td>
			<td style="width:123px;">Diatome&#160;</td>
			<td style="width:119px;">MC13437</td>
			<td style="width:177px;">equipped with a boat</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylenes</td>
			<td style="width:123px;">Fisher scientific</td>
			<td style="width:119px;">X5SK-4</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">150-mesh copper grids</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">G150-cu</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">grid-box</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">71138</td>
			<td style="width:177px;">Can store up to 100 grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium citrate</td>
			<td style="width:123px;">Anachemia</td>
			<td style="width:119px;">81983</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="336">
			<td height="336" style="height:336px;width:216px;">Lead nitrate</td>
			<td style="width:123px;">sigma</td>
			<td style="width:119px;">L-6258</td>
			<td style="width:177px;">Make a stock solution of lead citrate made of 1.33 g of lead nitrate and 1.76g of sodium citrate diluted in 42 mL of pre-boiled and cooled distilled water to which 8 mL of 1N NaOH are added after the conversion from lead nitrate to lead citrate is complete. pH should be approximately 12. Store solution in a hermetic plastic bottle and protect from light.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe</td>
			<td style="width:123px;">terumo&#160;</td>
			<td style="width:119px;">SS-05L</td>
			<td style="width:177px;">5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe filter</td>
			<td style="width:123px;">corning</td>
			<td style="width:119px;">431222</td>
			<td style="width:177px;">0.2 &#181;m</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Absorbing paper (bibulous paper)</td>
			<td style="width:123px;">Electron microscopic science</td>
			<td style="width:119px;">70086-1</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Parafilm</td>
			<td style="width:123px;">Laboratory film</td>
			<td style="width:119px;">PM-999</td>
			<td style="width:177px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mineral oil</td>
			<td style="width:123px;">Sigma</td>
			<td style="width:119px;">M5904</td>
			<td style="width:177px;"></td>
		</tr>
	</tbody>
</table>

preparation of non-human primate brain tissue for pre-embedding immunohistochemistry and electron microscopy

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and characterize ultrastructural and morphological details of neurons, such as synaptic contacts. Good preservation of brain tissue for electron microscopy can be obtained by rigorous cryo-fixation methods, but these techniques are rather costly and limit the use of immunolabeling, which is crucial to understand the connectivity of identified neuronal systems. Freeze-substitution methods have been developed to allow the combination of cryo-fixation with immunolabeling. However, the reproducibility of these methodological approaches usually relies on costly freezing devices. Moreover, achieving reliable results with this technique is very time-consuming and skill-challenging. Hence, the traditional chemically fixed brain, particularly with acrolein fixative, remains a time-efficient and low-cost method to combine electron microscopy with immunohistochemistry. Here, we provide a reliable experimental protocol using chemical acrolein fixation that leads to the preservation of primate brain tissue and is compatible with pre-embedding immunohistochemistry and transmission electron microscopic examination.

In this section, we present representative results that were obtained following the observation, at the transmission EM&#160;level, of immunostained primate brain tissue chemically fixed with a mixture of 3% acrolein and 4% PFA. We achieved good preservation of the ultrastructure, as indicated by the relatively intact myelin sheath and the neat visualization of double membranes (Figure 2A). Synaptic contacts, along with neuronal elements from the microenvironment, can eas...

Watch this Scientific Journal Video about Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy at JoVE.com

Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Despite all the technological advances at the light microscopy&#160;level, electron microscopy remains the only tool in neuroscience to examine and ...

preparation-non-human-primate-brain-tissue-for-pre-embedding

Research

JoVE Journal

Neuroscience

14.0K Views.  Université Laval. Here, we provide an easy, low-cost, and time-efficient protocol to chemically fix primate brain tissue with acrolein fixative, allowing for long-term preservation that is compatible with pre-embedding immunohistochemistry for transmission electron microscopy.

Video: Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy

Preparation of Mouse Brain Tissue for Immunoelectron Microscopy

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments (dendrite, dendritic spine, axon, axon terminal, perikaryon), as well as their intracellular organelles and cytoskeleton, in the central nervous system at high spatial resolution. Combined with TEM, pre-embedding immunocytochemistry allows the discrimination of cellular elements with few distinctive features and identification criteria (e.g., microglial perikarya and processes, when using an antibody against the microglia-specific marker Iba1 (ionized calcium binding adaptor molecule 1; as presented here)), identifying the neurotransmitter contents of cellular elements (e.g., serotonergic) and their ultrastructural localization of soluble or membrane-bound proteins (e.g., 5 HT1A and EphA4 receptors). Here, we describe a protocol for transcardiac perfusion of mice with acrolein fixative, removal and sectioning of the brain, as well as immunoperoxidase-diaminobenzidine (DAB) staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with TEM. When rigorously performed, this technique provides an excellent compromise between optimal ultrastructural preservation and immunocytochemical detection.

We describe a protocol for transcardiac perfusion of mice, removal and sectioning of the brain, as well as immunoperoxidase staining, resin embedding, and ultrathin sectioning of the brain sections. Upon completion of these procedures, the immunostained material is ready for examination with transmission electron microscopy.

Transmission electron microscopy (TEM) is extremely useful for visualizing microglial, oligodendrocytic, astrocytic, and neuronal subcellular compartments ...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

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 ...

Wholemount Immunohistochemistry for Revealing Complex Brain Topography

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its relatively uniform cytoarchitecture is a complex array of parasagittal domains of gene and protein expression. The molecular compartmentalization of the cerebellum is mirrored by the anatomical and functional organization of afferent fibers. To fully appreciate the complexity of cerebellar organization we previously refined a wholemount staining approach for high throughput analysis of patterning defects in the mouse cerebellum. This protocol describes in detail the reagents, tools, and practical steps that are useful to successfully reveal protein expression patterns in the adult mouse cerebellum by using wholemount immunostaining. The steps highlighted here demonstrate the utility of this method using the expression of zebrinII/aldolaseC as an example of how the fine topography of the brain can be revealed in its native three-dimensional conformation. Also described are adaptations to the protocol that allow for the visualization of protein expression in afferent projections and large cerebella for comparative studies of molecular topography. To illustrate these applications, data from afferent staining of the rat cerebellum are included.

Neural circuits are topographically organized into functional compartments with specific molecular profiles. Here, we provide the practical and technical steps for revealing global brain topography using a versatile wholemount immunohistochemical staining approach. We demonstrate the utility of the method using the well-understood cytoarchitecture and circuitry of cerebellum.

The repeated and well-understood cellular architecture of the cerebellum make it an ideal model system for exploring brain topography. Underlying its ...

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 ...

Primer for Immunohistochemistry on Cryosectioned Rat Brain Tissue: Example Staining for Microglia and Neurons

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brain tissue, using markers for microglia and neuronal elements as an example. Specifically, this paper is a step-by-step protocol for fluorescent visualization of microglia and neurons via immunohistochemistry for Iba1 and Pan-neuronal, respectively. Fluorescence double-labeling is particularly useful for the localization of multiple proteins within the same sample, providing the opportunity to accurately observe interactions between cell types, receptors, ligands, and/or the extracellular matrix in relation to one another as well as protein co-localization within a single cell. Unlike other visualization techniques, fluorescence immunohistochemistry staining intensity may decrease in the weeks to months following staining, unless appropriate precautions are taken. Despite this limitation, in many applications fluorescence double-labeling is preferred over alternatives such as 3,3&#39;-diaminobenzidine tetrahydrochloride (DAB) or alkaline phosphatase (AP), as fluorescence is more time efficient and allows for more precise differentiation between two or more markers. The discussion includes troubleshooting tips and advice to promote success.

This introductory level protocol describes the reagents, equipment, and techniques required to complete immunohistochemical staining of rodent brains, using markers for microglia and neuronal elements as an example.

Immunohistochemistry is a widely used technique for detecting the presence, location, and relative abundance of antigens in situ. This introductory level ...

Analysis of Brain Mitochondria Using Serial Block-Face Scanning Electron Microscopy

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, tri-carboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) pathways to produce cellular energy in the form of adenosine triphosphate (ATP). Impairment of mitochondrial ATP production causes mitochondrial disorders, which present clinically with prominent neurological and myopathic symptoms. Mitochondrial defects are also present in neurodevelopmental disorders (e.g. autism spectrum disorder) and neurodegenerative disorders (e.g. amyotrophic lateral sclerosis, Alzheimer&#39;s and Parkinson&#39;s diseases). Thus, there is an increased interest in the field for performing 3D analysis of mitochondrial morphology, structure and distribution under both healthy and disease states. The brain mitochondrial morphology is extremely diverse, with some mitochondria especially those in the synaptic region being in the range of &#60;200 nm diameter, which is below the resolution limit of traditional light microscopy. Expressing a mitochondrially-targeted green fluorescent protein (GFP) in the brain significantly enhances the organellar detection by confocal microscopy. However, it does not overcome the constraints on the sensitivity of detection of relatively small sized mitochondria without oversaturating the images of large sized mitochondria. While serial transmission electron microscopy has been successfully used to characterize mitochondria at the neuronal synapse, this technique is extremely time-consuming especially when comparing multiple samples. The serial block-face scanning electron microscopy (SBFSEM) technique involves an automated process of sectioning, imaging blocks of tissue and data acquisition. Here, we provide a protocol to perform SBFSEM of a defined region from rodent brain to rapidly reconstruct and visualize mitochondrial morphology. This technique could also be used to provide accurate information on mitochondrial number, volume, size and distribution in a defined brain region. Since the obtained image resolution is high (typically under 10 nm) any gross mitochondrial morphological defects may also be detected.

Mitochondrial visualization and analysis from mammalian brain tissue is a challenging task. Here, we describe how three dimensional (3D) reconstruction analysis from the serial block-face scanning electron microscopy (SBFSEM) can be used to gain insights on the morphological and volumetric analysis of this critical energy generating organelle.

Human brain is a high energy consuming organ that mainly relies on glucose as a fuel source. Glucose is catabolized by brain mitochondria via glycolysis, ...

Preparation of Mouse Retinal Cryo-sections for Immunohistochemistry

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for determining the mechanisms underlying retinal diseases. Maintaining structural integrity throughout the tissue preparation is vital for obtaining reproducible retinal IHC data but can be challenging due to the fragility and complexity of retinal cytoarchitecture. Strong fixatives like 10% formalin or Bouin's solution optimally preserve the retinal structure, they often impede IHC analysis by enhancing the background fluorescence and/or diminishing antibody-epitope interactions, a process known as epitope masking. Milder fixatives, on the other hand, like 4% paraformaldehyde, reduces background fluorescence and epitope-masking, meticulous dissection techniques must be utilized to preserve the retinal structure. In this article, we present a comprehensive method to prepare mouse ocular posterior cups for IHC that is sufficient to preserve most antibody-epitope interactions without loss of retinal structural integrity. We include representative IHC with antibodies to various retinal cell type markers to illustrate tissue preservation and orientation under optimal and sub-optimal conditions. Our goal is to optimize IHC studies of the retina by providing a complete protocol from ocular posterior cup dissection to IHC.

This report describes comprehensive methods for preparing frozen mouse retina sections for immunohistochemistry (IHC). Methods described include dissection of the ocular posterior cup, paraformaldehyde fixation, embedding in Optimal Cutting Temperature (OCT) media and tissue orientation, sectioning and immunostaining.

Preparation of high-quality mouse eye sections for immunohistochemistry (IHC) is critical for assessing the retinal structure and function and for ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

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 ...