eoe sub articles

EoE Sub Articles

1. Animal preparation
<ol>
	<li>Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen and pulling the skin. Carefully make another incision to open the chest cavity without puncturing any other organs.
	<ol>
		<li>Perfuse&#160;the heart through the apex first and then the right ventricle using cold 3% glutaraldehyde in cardioplegic solution (50 mM KCl, 5% dextrose in PBS, see&#160;Table 1) for 2 min to ensure complete myocardial relaxation.</li>
	</ol>
	</li>
	<li>Perfuse the heart using ice-cold 3% glutaraldehyde in 0.1 M of sodium cacodylate buffer (pH 7.4) for another 2 min. Using gravity pressure, use a 25 G 5/8&#34; needle to introduce the fixatives into the heart. Immediately after the heart starts to fill up with the fixative, lift up the apex of the heart and cut the vessels underneath at 1-2 mm from the heart to relieve pressure and allow the liquids to drain.</li>
	<li>Dissect the heart, remove the atria, and drop the ventricles into ice-cold 3% glutaraldehyde/0.1 M sodium cacodylate in a Petri dish. Make a butterfly cut after 30-60 min of fixation and place it in a Petri dish containing 3% glutaraldehyde/cacodylate (see&#160;Table of Materials). Chop the heart using a surgical blade in small cubes of 1 mm3&#160;while it is in glutaraldehyde/cacodylate solution.</li>
	<li>Fix/immerse the dissected heart tissue in glutaraldehyde/cacodylate solution for 24 h at 4 &#176;C.</li>
</ol>
2. Heart tissue processing
<ol>
	<li>After 24 h of fixation, wash the tissues in 0.1 M of sodium cacodylate buffer for 20 min.
	<ol>
		<li>Prepare 0.1 M cacodylate buffer following the steps below.
		<ol>
			<li>To prepare 0.1 M sodium cacodylate buffer, prepare a stock of 1 M sodium cacodylate buffer by dissolving sodium cacodylate (21.4 g) and 1% calcium chloride solution (3 mL) in 90 mL of distilled water. Volume up the solution to 100 mL by adding the distilled water. Stir the solution well and leave it overnight to dissolve the solute.</li>
			<li>Next, take 10 mL of 1 M sodium cacodylate stock solution and add it to 80 mL of distilled water. Adjust the pH to 7.4 using HCl and volume it up to 100 mL by adding distilled water.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Repeat the wash in 0.1 M sodium cacodylate buffer for another 20 min. Remove the tissues from sodium cacodylate buffer and immerse them in 2% osmium tetroxide solution for 4 h at room temperature. This process is called osmication.
	<ol>
		<li>Prepare 2% Osmium tetroxide solution following the steps below.
		<ol>
			<li>To prepare 2% Osmium tetroxide solution, take 4% Osmium tetroxide solution, 4 mL (see Table of Materials), 1 M sodium cacodylate stock solution (0.8 mL), and distilled water (3.2 mL) to make a total of 8 mL solution. 
			NOTE: This whole process and the steps from hereon must be carried out in the fume hood.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>After osmication, immerse the tissues in 2% sodium acetate solution for 10 min at room temperature.
	<ol>
		<li>Prepare 2% sodium acetate solution by dissolving 4 g of sodium acetate in 20 mL of distilled water.</li>
	</ol>
	</li>
	<li>Next, immerse the tissues in 2% uranyl acetate solution for 1 h at room temperature.
	<ol>
		<li>Prepare 2% uranyl acetate solution by dissolving 4 g of uranyl acetate in 20 mL of distilled water.</li>
	</ol>
	</li>
	<li>After uranyl acetate staining, dehydrate the tissues sequentially through the graded alcohols and acetone in the order mentioned in Table 2.</li>
</ol>
3. Heart tissue embedding
<ol>
	<li>Embed the dehydrated tissues in low-viscosity epoxy resin following the steps below.
	<ol>
		<li>To make low-viscosity epoxy resin, mix 10.24 mL of vinyl cyclohexene dioxide epoxy monomer, 6.74 mL of diglycidyl ether of polypropylene glycol (DER 732), and 30.05 mL of nonenyl succinic anhydride (NSA) (see Table of Materials) in a 50 mL tube. Stir the suspension well by hand for 2 min.</li>
		<li>Add 18 drops of 2-dimethylaminoethanol (DMAE, see Table of Materials) epoxy accelerator and stir the suspension to mix the components thoroughly.</li>
		<li>Impregnate the tissues in epoxy resin in the following sequence of mixture.
		<ol>
			<li>Replace the 100% acetone with 1:1 resin: acetone suspension and immerse the tissues in it for 1 h at room temperature.</li>
			<li>Replace 1:1 resin: acetone suspension with 6:1 resin: acetone suspension and immerse the tissues in it for 3 h.</li>
			<li>Finally, replace the 6:1 resin: acetone suspension with 100% resin suspension and leave tissues immersed in it overnight at room temperature.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Put the tissues into fresh resin in 8 mm micro molds (see Table of Materials) and cure the embedded tissues at 70 &#176;C overnight. 
	NOTE: Ensure that the resin is hard but not brittle after curing.</li>
</ol>
4. Tissue sectioning using an ultramicrotome
<ol>
	<li>Trim the resin blocks with the tissue to no larger than 1 mm before mounting on the ultramicrotome (see Table of Materials). Place the mold as precisely as possible in the segmented arm of the ultramicrotome and manually advance the sample mold toward the diamond knife.</li>
	<li>Cut the sections at a thickness of 500 nm (one-half micron) with a Histo knife (see Table of Materials), and using the EM loop tool, pick them up and place them on a glass slide.</li>
	<li>Place the glass slide on a hot plate for toluidine blue stain to find the area of interest.</li>
	<li>After finding the area of interest, use an Ultra 45&#176; knife (see Table of Materials) to produce pale gold ultrathin sections (100 nm). Place these ultrathin sections on the dull side of a 200 mesh copper grid.</li>
</ol>
5. Ultrathin heart section staining
<ol>
	<li>Start the staining by unmasking the antigen using an etching solution, i.e., 5% sodium meta-periodate solution.
	<ol>
		<li>Prepare 500 &#181;L of 5% sodium metaperiodate (see Table of Materials) solution in distilled water. 
		NOTE: Prepare fresh solution before use.</li>
		<li>Put 20 &#181;L of sodium metaperiodate solution on a clean paraffin film. Place the completely dried grids with tissue sections on the droplet of the etching solution. 
		NOTE: The dull side of the grid with the tissue section must face toward the etching solution.</li>
		<li>Leave the section grid on the solution for 30 min at room temperature.</li>
	</ol>
	</li>
	<li>Wash the etched tissue section by placing it on a droplet of distilled water for 60 s.</li>
	<li>Block the residual aldehydes by placing the section grids on a droplet of 0.05 M glycine solution for 10 min at room temperature. Blot the grid&#39;s edges on filter paper to remove the residual glycine solution.
	<ol>
		<li>Prepare 0.05 M glycine solution (see Table of Materials) by dissolving 3.75 mg of glycine in 1 mL of 1x PBS (pH 7.4).</li>
	</ol>
	</li>
	<li>Place the section grids in 10-20 &#181;L of blocking solution for 25 min at room temperature. 
	NOTE: Blocking solution composition: 2 &#181;L of 1% normal goat serum (NGS) + 20 &#181;L of 1% BSA (final concentration: 10%) + 178 &#181;L of 1x PBS (pH 7.4) to make a final volume of 200 &#181;L.</li>
	<li>Blot the grid edges on filter paper and place the grid section on antibody diluent for conditioning at room temperature for 10 min.</li>
	<li>Incubate the grid sections with primary antibody (Sigmar1 in this case, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h 30 min in a humidified chamber.</li>
	<li>Blot dry the grid and wash the grid sections with antibody diluent twice for 5 min each.</li>
	<li>Incubate the grid sections with biotinylated secondary antibody (in this case, biotinylated goat anti-rabbit polyclonal secondary antibody, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h in a humidified chamber.</li>
	<li>Blot dry the grid and wash the grid sections with antibody diluent twice for 5 min each.</li>
	<li>Incubate the grid sections in commercially available streptavidin-conjugated QD (QD655nm, see Table of Materials; diluted 1:10 in antibody diluent) for 1 h in a humidified chamber at room temperature. Prevent exposure to light by covering the chamber with aluminum foil.</li>
	<li>Blot dry the grid edges using filter paper and wash the grid sections by placing them on water droplets for 2 min.</li>
	<li>Blot the edges of the grid to dry.</li>
</ol>
Table 1: Composition of PBS and other solutions used for tissue dehydration.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Buffer</td>
			<td>Composition</td>
			<td>Quantity</td>
		</tr>
		<tr>
			<td rowspan="5">Phosphate-buffered Saline (PBS) (1x) (1 L)</td>
			<td>Sodium Chloride</td>
			<td>8 g</td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>0.2 g</td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic</td>
			<td>1.44 g</td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic</td>
			<td>0.24 g</td>
		</tr>
		<tr>
			<td>Adjust the pH to 7.4</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Application of graded alcohol and acetone for tissue dehydration (Step 2.5)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Component</td>
			<td>Time</td>
		</tr>
		<tr>
			<td>50% ethanol</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>95% ethanol</td>
			<td>10 min</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>20 min</td>
		</tr>
		<tr>
			<td>100% ethanol</td>
			<td>20 min</td>
		</tr>
		<tr>
			<td>100% acetone</td>
			<td>30 min</td>
		</tr>
		<tr>
			<td>100% acetone</td>
			<td>30 min</td>
		</tr>
	</tbody>
</table>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>200 Mesh copper grid</td>
			<td>Ted Pella</td>
			<td>G200HH</td>
			<td></td>
		</tr>
		<tr>
			<td>6-month-old male mice with FVB/N background</td>
			<td>Jackson Laboratory, Bar Harbor, ME</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone 100%</td>
			<td>Fisher Chemicals</td>
			<td>A949</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody diluent</td>
			<td>Dako</td>
			<td>S3022</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Sigmar1 antibody</td>
			<td>Cell Signaling</td>
			<td>61994S</td>
			<td></td>
		</tr>
		<tr>
			<td>Biotinylated goat anti-rabbit IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>B7389</td>
			<td></td>
		</tr>
		<tr>
			<td>BSAc (10%)</td>
			<td>Electron Microscopy Sciences</td>
			<td>25557</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma Aldrich</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>Cytoseal Xyl</td>
			<td>Thermo fisher</td>
			<td>8312-4</td>
			<td></td>
		</tr>
		<tr>
			<td>DER 732C36AA10:C33</td>
			<td>Electron Microscopy Sciences</td>
			<td>13010</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Knife</td>
			<td>Diatome</td>
			<td>Histo; Ultra 450</td>
			<td></td>
		</tr>
		<tr>
			<td>DMAE</td>
			<td>Electron Microscopy Sciences</td>
			<td>13300</td>
			<td></td>
		</tr>
		<tr>
			<td>Electron microscope</td>
			<td>JEOL</td>
			<td>JEOL-1400 Flash</td>
			<td></td>
		</tr>
		<tr>
			<td>ERL 4221</td>
			<td>Electron Microscopy Sciences</td>
			<td>15004</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 100%</td>
			<td>Fisher Chemicals</td>
			<td>A405P</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde 3%</td>
			<td>Electron Microscopy Sciences</td>
			<td>16538-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Alfa Aesar</td>
			<td>A13816</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Fisher Scientific</td>
			<td>SA56</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromolds</td>
			<td>Ted Pella</td>
			<td>10505</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Microsystem</td>
			<td>EM UC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr>
			<td>NSA</td>
			<td>Electron Microscopy Sciences</td>
			<td>19050</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetroxide</td>
			<td>Electron Microscopy Sciences</td>
			<td>19150</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Genesse Scientific</td>
			<td>16-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>71640</td>
			<td></td>
		</tr>
		<tr>
			<td>Qdot 655 Streptavidin Conjugate</td>
			<td>Invitrogen</td>
			<td>Q10121MP</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate</td>
			<td>Fisher Scientific</td>
			<td>BP334</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Cacodylate</td>
			<td>Electron Microscopy Sciences</td>
			<td>12300</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium metaperiodate</td>
			<td>Sigma Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Phosphate dibasic</td>
			<td>Sigma Aldrich</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical blade (size 10)</td>
			<td>Aspen surgical</td>
			<td>371110</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM image software</td>
			<td>AMT-V700</td>
			<td>AMT TEM imaging systems</td>
			<td></td>
		</tr>
		<tr>
			<td>TEM imaging camera</td>
			<td>XR80 TEM series</td>
			<td>AMT TEM imaging systems</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue O solution (0.5%)</td>
			<td>Fisher Scientic</td>
			<td>S25612</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Polysciences</td>
			<td>21447</td>
			<td></td>
		</tr>
	</tbody>
</table>

an immunolabeling technique to visualize the subcellular localization of proteins

Source: Aishwarya, R., et al. <a href="https://app-jove-com.remotexs.ntu.edu.sg/v/64085">Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy.</a> J. Vis. Exp. (2022).
This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with quantum dots to demarcate the subcellular localization of Sigma-1 receptors using electron microscopy.

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

1. Animal preparation

	Anesthetize the mice using 3% isoflurane. Open the chest by making a horizontal incision in the region above the middle abdomen ...

Begin by washing a fixed heart section in a sodium cacodylate buffer that stabilizes the tissue structure. Immerse the section in osmium tetraoxide to stain the membrane lipids.
Treat with sodium acetate to maintain an optimum pH for staining.
Add uranyl acetate to stain biomolecules, providing better contrast during microscopy.
Dehydrate the tissue in increasing concentrations of ethanol followed by acetone.
Embed the tissue in a resin, obtain ultrathin sections, and transfer them onto a microscopy grid.
Introduce sodium meta-periodate to remove the osmium tetraoxide, unmasking the cellular protein.&#160;
The compound also oxidizes glycoprotein, creating reactive aldehyde groups, which are neutralized by glycine treatment.
Introduce a blocking buffer, preventing non-specific antibody binding.
Add primary antibodies specific to the target protein followed by biotinylated secondary antibodies.
Add streptavidin-conjugated quantum dots that bind to biotin.
Under an electron microscope, quantum dot-labeled regions exhibit higher contrast, aiding precise subcellular localization of the target protein.

an-immunolabeling-technique-to-visualize-subcellular-localization

Nanyang Technological University

Research

JoVE Encyclopedia of Experiments

Immunology

Antibody-Based Technologies

Antibody-Based Imaging and Detection Methods

101 Views. Source: Aishwarya, R., et al. Visualizing Subcellular Localization of a Protein in the Heart Using Quantum Dots-Mediated Immuno-Labeling Followed by Transmission Electron Microscopy. J. Vis. Exp. (2022). This video demonstrates a technique for the immunolabeling of sigma-1 receptors in a murine heart tissue section using quantum dots. The heart tissue is fixed, osmicated, stained, dehydrated, embedded in resin, and then sectioned. The sections are then treated with antibodies and labeled with q...

Video: An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins - Concept

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

JoVE Journal

Biology

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum dots are fluorescent nanoparticles with excellent photophysical properties imbued with bright and stable photoluminescence as well as narrow emission bands. Quantum dots are spherical in shape, and with the proper modification of the surface chemistry, can be used to conjugate biomolecules for cellular applications. These optical properties, combined with the ability to functionalize them with biomolecules, make them an excellent tool for investigating receptor-ligand interactions and cellular trafficking. Here, we present a method that uses quantum dots to track the binding and endocytosis of SARS-CoV-2 spike protein. This protocol can be used as a guide for experimentalists looking to utilize quantum dots to study protein-protein interactions and trafficking in the context of cellular physiology.

In this protocol, quantum dots conjugated to recombinant SARS-CoV-2 spike enable cell-based assays to monitor spike binding to hACE2 at the plasma membrane and subsequent endocytosis of the bound proteins into the cytoplasm.

The development of new technologies for cellular fluorescence microscopy has facilitated high-throughput screening methods for drug discovery. Quantum ...

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Transcript

An Immunolabeling Technique to Visualize the Subcellular Localization of Proteins

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

High-throughput Confocal Imaging of Quantum Dot-Conjugated SARS-CoV-2 Spike Trimers to Track Binding and Endocytosis in HEK293T Cells