Name: preparation of non-human primate brain tissue for pre-embedding immunohistochemistry and electron microscopy
Uploaded: 2017-04-03T13:00:00
Description: Watch this Scientific Journal Video about Preparation of Non-human Primate Brain Tissue for Pre-embedding Immunohistochemistry and Electron Microscopy at JoVE.com

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
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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 ...

The overall goal of this experiment is to obtain reliable pre-embedding immunostaining of non-primate brain tissue for electron microscopy. This method can help answer key questions in the neuroanatomy field, such as the synaptic incidence of a given innervation in a precise brain structure. The main advantage of this technique is that it's an easy and low cost method to label specific innervations suitable for electron microscopy.Begin by transferring 50 micron thick sections of acrolein fixed non-human primate brain containing the region of interest to a 12 well plate containing PBS. Wash the free floating sections three times in PBS for five minutes at room temperature, then incubate the sections in freshly prepared sodium borohydride solution for 30 minutes at room temperature. Rock gently without a cover.Once the 30 minutes have elapsed, aspirate the sodium borohydride and add PBS to each well. Place the plate on a rocker and shake vigorously for 10 minutes. Repeat the wash two more times or until none of the reaction gas remains.Block the sections by incubating in a solution of 2%normal serum and 0.5%cold fish gelatin diluted in PBS for one hour at room temperature with gentle rocking. Once the incubation time has elapsed, incubate the sections in primary antibody solution with gentle rocking overnight at room temperature. The next day, after washing the sections as before, incubate the sections in secondary antibody solution for one and a half hours at room temperature with gentle rocking.After washing the sections as before, incubate in avidin-biotin-peroxidase or ABC solution for one hour with rocking. Next, prepare a fresh solution of 0.05%3, 3'Diaminobenzidine with 0.005%hydrogen peroxide diluted in TBS. After washing, incubate sections in the DAB solution for three to seven minutes with gentle rocking.Once the brown precipitate has developed to the desired color, stop the reaction by quickly washing twice in cold TBS, then through a series of timed TBS and PB washes. Transfer the plate containing the DAB-stained sections to the venting hood. Transfer sections into a six well plate filled with PB and completely flatten the sections by carefully pipetting the PB out of the plate, then add osmium tetroxide solution drop by drop to avoid disturbing the flattened sections.Cover the plate with aluminum foil to protect the sections from light and incubate for 30 minutes at room temperature without agitation. During the osmification, prepare water repellent epoxy resin by adding appropriate amounts of each component of the epoxy resin mix to a large plastic cup. Stir with a wooden stick or plastic pipette until a homogeneous brown color is obtained, then transfer equal quantities of resin to aluminum cups of appropriate size for the number of sections to be processed and allow it to rest.Once the osmification period has elapsed, wash the sections three times in PB for 10 minutes with low speed rocking. After washing, dehydrate the sections in the following series of graded ethanol for two minutes each. Next, transfer the sections to glass vials filled with propylene oxide and incubate in propylene oxide three times for two minutes each to complete the dehydration process.Then transfer the sections carefully, one by one, into the aluminum cups, avoiding 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 room temperature. The next day, coat glass slides and plastic cover slips with mineral oil, then soften the resin by incubating the aluminum cups at 60 degrees Celsius for 12 to 15 minutes at most.Carefully flatten the sections on the greased side of the glass slide. Place the greased cover slip and carefully push out any remaining air. Incubate the slides at 60 degrees Celsius for 48 hours.After 48 hours, remove the plastic cover slip, leaving the section encased in resin. Begin by using binoculars to locate the region of interest and then use a scalpel to cut a small quadrangular piece of tissue of approximately one milliliter squared. Next, file the tip of a resin block and then glue the quadrangular tissue piece onto it.After drying, place the resin block in the ultramicrotome deck in the vertical position and use a sharp razor blade to gradually cut each side of the resin block to form a trapezoid with smooth sides. Next, put the deck in its horizontal position and rotate the block until the longest side of the trapezoid is facing downward. Place a diamond trimming tool or a glass knife in dedicated space.Set the ultramicrotome to cut 300 micron sections at one millimeter per second. Adjust the knife to be vertically parallel to the quadrangular piece and to display a very small horizontal angle of approximately one degree and trim the surface of the quadrangular piece until the tissue starts to appear. Now, switch to an ultra 45 degree diamond knife equipped with a boat filled with distilled water to cut 80 micron thick sections.Smoothen sections by going over them with a piece of absorbing paper tipped in xylene without touching the surface of the water, and collect serial sections on bare 150 mesh copper grids. Place the grids in a grid storage box then prepare a one to one solution of lead citrate stock solution and distilled water and filter through a 0.2 micron syringe filter. Cover the vial with aluminum foil to protect it from light.Place each grid onto a drop of the diluted lead citrate solution, with the section in contact with the solution Then use small tweezers to hold the grid and thoroughly rinse it in two beakers containing distilled water. Gently remove the excess water with absorbing paper and place the grids in a grid box. After 30 minutes, the sections may be examined by transmission electron microscopy.This electron micrograph of the squirrel monkey internal globus pallidus shows well-preserved material after acrolein-PFA transcardiac perfusion in the immunoperoxidase diaminobenzidine technique. As seen there, the procedure allows the myelin sheath of a large axon, indicated by the arrowhead, to be seen. This electron micrograph of the external globus pallidus demonstrates the dendritic profiles, indicated by the letter d, small unmyelinated axons, indicated by a, and axon varicosities, labeled av, can easily be identified.The arrows show an example of an axon varicosity establishing a symmetrical synaptic contact with the dendritic profile. Immunolabeled elements can easily be identified by their cytoplasm or axoplasm filled with electron dense DAB precipitate. Here, a dendrite immunostained for choline acetyltransferase in the GPi receives a synaptic contact from an unlabeled axon varicosity.This electron micrograph shows a myelinated axon in the GPe with a relatively intact myelin sheath whose axoplasm is immunolabeled for tyrosine hydroxylase. This example shows an axon varicosity in the GPi immunolabeled for serotonin transporter that establishes a symmetrical synaptic contact with a dendrite. In this example, the DAB precipitate lines the plasma membrane and the outer surface of organelles.The axon varicosity shown here was observed in the GPe and immunolabeled for tyrosine hydroxylase. This represents an example of DAB precipitate entirely filling the axoplasm, with synaptic vesicles being visible but more difficult to delineate. Following this procedure, other methods, like pre-embedding double immunohistochemistry can be performed to answer additional questions like colocalizations of proteins or receptors at the synaptic level.After watching this video, you should have a good understanding of how to prepare pre-embedding immunohistochemistry of non-primate brain tissue, proceed to the embedding in epoxy resin, and ultracut the region of interest into 80 micrometer thick sections suitable for electron microscopy.

Research

JoVE Journal

Neuroscience

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