Name: An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis (Video) | JoVE Journal
Uploaded: 2016-10-12T15:00:00.000+00:00
Duration: 9 min 17 s
Description: Learn an alternative TEM sample preparation method for low cell numbers using hematopoietic stem cells. This efficient protocol enables reproducible visualization of cellular ultrastructure in biomedical research.

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5 minutes

We thank the Pathology Research Core for assistance with electron microscope analysis studies at Cincinnati Children's Hospital Medical Center. The work was supported by NIH (American Society of Hematology Bridge award to-MDF; R01 DK102890 to MDF).

All experimental procedures were approved by the institutional animal committee at the Cincinnati Children&#39;s Research Foundation. For this study, hematopoietic stem cells were isolated from the bone marrow of C57Bl/6 inbred mice aged 2 - 4 months. Cell sorting using FACS after staining of BM with different surface makers including Lineage, c-Kit, Sca-1, Flt3 and CD34 was used for purification of HSC based on Lin- Sca-1+ c-Kit+ Flt3- CD34- gating strategy as standard protocol described before8.
CAUTION: Several highly toxic chemicals are used during this procedure. These are toxic by inhalation and skin contact. Please wear gloves and protective clothing. Work in fume hood while working with these chemicals.
1. Cells, Fixation, Staining and Pre-embedding
<ol>
	<li>Sort 10,000 hematopoietic stem cells (using Lin- Sca-1+ c-Kit+ Flt3- CD34- gating strategy; LT-HSC population) in a 1.5 ml microcentrifuge tube containing 600 &#181;l Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM) with 10% fetal bovine serum using FACS.</li>
	<li>Spin down sorted HSCs at 1,000 x g for 10 min in a 1.5 ml microcentrifuge tube using a centrifuge with swing-out buckets. Remove medium by gentle aspiration and leave 200 &#181;l medium in the tubes at each step. At this stage, the cell pellet remains invisible in the tube.</li>
	<li>Fix the cells by adding 0.2 ml of 2x fixative solution at room temperature (RT) for 1 hr1,9.
	<ol>
		<li>Make the fixative solution fresh before use. At 1x, the solution is comprised of 3% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer.</li>
	</ol>
	</li>
	<li>Centrifuge the cells at 1,000 x g for 10 min at 30 &#176;C using a centrifuge with swing-out buckets.</li>
	<li>Wash cells with 600 &#181;l of 0.1 M cacodylate buffer using centrifugation and leave 200 &#181;l buffer in the tube after centrifugation.</li>
	<li>Stain the fixed cells by adding 200 &#181;l of 2 mg/ml solution of Evans Blue in cacodylate buffer and incubate for 20 min at RT. The cells will be colored blue.</li>
	<li>Wash cells 3 times with 600 &#181;l of 0.1 M cacodylate buffer using centrifugation and leave 200 &#181;l buffer in the tube each time.</li>
	<li>Re-suspend the cells in 200 &#181;l buffer and transfer the cell suspension to a 0.5 ml tube. Centrifuge at 1,000 x g for 10 min using a table top centrifuge. Remove buffer gently without disturbing the now visible tiny cell pellet, and leave 50 &#181;l buffer in the tube.</li>
	<li>Add 200 mg low melting agarose to 5 ml of 0.1 M cacodylate buffer in the 15 ml tube for a 4% agarose solution. Melt the agarose by transferring the tube to boiling water in a 100 ml glass bottle and keep lukewarm until used.</li>
	<li>Add 200 &#181;l of 4% low melting agarose dissolved in 0.1 M cacodylate buffer to the cell suspension in 0.5 ml tube and immediately centrifuge at 1,000 x g for 10 min at 30 &#176;C using a table top centrifuge.</li>
	<li>After confirming that cells pelleted at bottom of the 0.5 ml tube into the semi-solid agarose after centrifugation, immediately transfer the tube to 4 &#176;C or ice for 20 min to solidify the agarose. This step is a very critical, as a small visible cell pellet at this step results in a good cluster of cells under the electron microscope.</li>
	<li>Carefully transfer the solidified agarose containing the cell pellet from the tube to a 35 mm plastic Petri dish containing buffer using a 27 G needle3,10.</li>
	<li>Trim the solidified agarose containing the cell pellet into one piece of about 1 - 2 mm with a scalpel. Transfer the agarose piece containing the cell pellet to a new 1.5 ml tube.</li>
	<li>Wash 2 - 3 times using 600 &#181;l of 0.1 M cacodylate buffer by adding and removing buffer using pipette without centrifugation. Use this agarose piece containing the cell pellet for the next step. At this step, the sample can be stored at 4 &#176;C overnight before moving to next step.</li>
</ol>
2. Osmification, Dehydration, Embedding
<ol>
	<li>Remove the cacodylate buffer from the 1.5 ml tube with the cell pellet containing agarose using a pipette. Add 1.0 ml of 1% osmium tetraoxide (OsO4) solution in a fume hood, and incubate for 1 hr at 4 &#176;C.</li>
	<li>Wash three times with 600 &#181;l of 0.1 M cacodylate buffer and transfer the sample to a 20 ml glass scintillation vial with a cap.</li>
	<li>Process the sample for dehydration, infiltration and embedding into resin (e.g., LX-112) with serial changes of the following solutions in a 6 well plate. Move the cell-pellet agarose piece using forceps.
	<ol>
		<li>Immerse the sample in 25% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 50% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 75% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 95% ethanol at RT for 15 min.</li>
		<li>Immerse the sample in 100% ethanol at RT for 15 min, twice.</li>
		<li>Immerse the sample in ethanol: resin (3:1) at RT for 30 min.</li>
		<li>Immerse the sample in ethanol: resin (1:1) at RT for 30 min.</li>
		<li>Immerse the sample in ethanol: resin (1:3) at RT for 30 min.</li>
		<li>Immerse the sample in pure resin at RT for 60 min, twice.</li>
	</ol>
	</li>
	<li>Transfer the sample to the bottom of pyramid tip shaped rubber mold using forceps carefully and add more pure resin on top of the sample to fill the mold. Incubate the sample at 60 &#176;C for 72 hr for resin polymerization.</li>
	<li>Remove the polymerized resin pyramid from the molds by twisting the rubber mold. A small black cluster of cells should be visible in the polymerized resin pyramid, as described earlier7. Attach the polymerized resin pyramid on the mounting cylinder using cyanoacrylate.</li>
</ol>
3. Sectioning and Transmission Electron Microscopy
<ol>
	<li>Trim the pyramid block with a razor blade and a diamond knife after mounting the block on an ultra-microtome. Make a short wide trapezoidal shape with the top and the bottom of block parallel to each other, and with evenly angled sides. This shape of the pyramid helps for serial sectioning.</li>
	<li>Further, trim the block around the cell pellet with the diamond knife and remove the plastic sections around the cell pellet agarose piece.</li>
	<li>Cut 1 &#181;m sections using an ultra-microtome. Move the 1 &#181;m sections from the water boat to a glass slide using an eyelash tool and a metal loop tool as previously reported7. Transfer sections on slides and transfer slides to the hot plate (37 &#176;C) for drying.</li>
	<li>Add a drop of toluidine blue solution to the sections using a syringe equipped with 0.22 &#181;m filter and incubate for 3 min. Rinse the slides with distilled water and let the sections dry. Check with a light microscope to identify the position of the cells.</li>
	<li>Cut ultra-thin sections (70 - 100 nm) using a diamond knife. Move 2 - 3 sections on 200-mesh grids from the water boat to a glass Petri dish using an eyelash tool.</li>
	<li>Transfer the glass Petri dish containing grids with sections to a hot plate at 30 &#176;C to dry sections for 30 min.</li>
	<li>Stain the grids with drops of 1% uranyl acetate at RT for 10 min and then rinse in distilled water 6 - 8 times. Stain with Reynolds lead citrate, at RT for 5 min and rinse in distilled water 6 - 8 times. Transfer the glass Petri dish containing the grids to a hot plate at 37 &#176;C to dry sections.</li>
	<li>Analyze the sections with an electron microscope, at 80 kV7.</li>
</ol>

<ol><li>Bozzola, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Conventional+specimen+preparation+techniques+for+transmission+electron+microscopy+of+cultured+cells%5BTitle%5D)+AND+%22Methods+Mol+Biol%22%5BJournal%5D)">Conventional specimen preparation techniques for transmission electron microscopy of cultured cells.</a> Methods Mol Biol. 369, 1-18 (2007).</li>
<li>Leapman, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Novel+techniques+in+electron+microscopy%5BTitle%5D)+AND+%22Curr+Opin+Neurobiol%22%5BJournal%5D)">Novel techniques in electron microscopy.</a> Curr Opin Neurobiol. 14, 591-598 (2004).</li>
<li>Anderson, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+method+of+preparing+peripheral+leucocytes+for+electron+microscopy%5BTitle%5D)+AND+%22J+Ulstract+Res%22%5BJournal%5D)">A method of preparing peripheral leucocytes for electron microscopy.</a> J Ulstract Res. 13, 263-268 (1965).</li>
<li>Taupin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Processing+scarce+biological+samples+for+light+and+transmission+electron+microscopy%5BTitle%5D)+AND+%22Eur+J+Histochem%22%5BJournal%5D)">Processing scarce biological samples for light and transmission electron microscopy.</a> Eur J Histochem. 52, 135-139 (2008).</li>
<li>Mather, J., Stanbridge, C. M., Butler, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Method+for+the+removal+of+selected+cells+from+cytological+smear+preparations+for+transmission+electron+microscopy%5BTitle%5D)+AND+%22J+Clin+Pathol%22%5BJournal%5D)">Method for the removal of selected cells from cytological smear preparations for transmission electron microscopy.</a> J Clin Pathol. 34, 1355-1357 (1981).</li>
<li>Oorschot, V., de Wit, H., Annaert, W. G., Klumperman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+flat-embedding+method+to+prepare+ultrathin+cryosections+from+cultured+cells+in+their+in+situ+orientation%5BTitle%5D)+AND+%22J+Histochem+Cytochem%22%5BJournal%5D)">A novel flat-embedding method to prepare ultrathin cryosections from cultured cells in their in situ orientation.</a> J Histochem Cytochem. 50, 1067-1080 (2002).</li>
<li>Kumar, S., Ciraolo, G., Hinge, A., Filippi, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+efficient+and+reproducible+process+for+transmission+electron+microscopy+%28TEM%29+of+rare+cell+populations%5BTitle%5D)+AND+%22J+Immunol+Methods%22%5BJournal%5D)">An efficient and reproducible process for transmission electron microscopy (TEM) of rare cell populations.</a> J Immunol Methods. 404, 87-90 (2014).</li>
<li>Chen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Enrichment+of+hematopoietic+stem+cells+with+SLAM+and+LSK+markers+for+the+detection+of+hematopoietic+stem+cell+function+in+normal+and+Trp53+null+mice%5BTitle%5D)+AND+%22Exp+Hematol%22%5BJournal%5D)">Enrichment of hematopoietic stem cells with SLAM and LSK markers for the detection of hematopoietic stem cell function in normal and Trp53 null mice.</a> Exp Hematol. 36, 1236-1243 (2008).</li>
<li>Hopwood, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+reactions+between+formaldehyde%2C+glutaraldehyde+and+osmium+tetroxide%2C+and+their+fixation+effects+o+bovine+serum+albumin+and+on+tissue+blocks%5BTitle%5D)+AND+%22Histochemistry%22%5BJournal%5D)">The reactions between formaldehyde, glutaraldehyde and osmium tetroxide, and their fixation effects o bovine serum albumin and on tissue blocks.</a> Histochemistry. 24, 50-64 (1970).</li>
<li>Saini, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Nitric+oxide+synthase+localization+in+the+rat+neutrophils%3A+immunocytochemical%2C+molecular%2C+and+biochemical+studies%5BTitle%5D)+AND+%22J+Leukoc+Biol%22%5BJournal%5D)">Nitric oxide synthase localization in the rat neutrophils: immunocytochemical, molecular, and biochemical studies.</a> J Leukoc Biol. 79, 519-528 (2006).</li>
<li>Kumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+small+GTPase+Rap1b+negatively+regulates+neutrophil+chemotaxis+and+transcellular+diapedesis+by+inhibiting+Akt+activation%5BTitle%5D)+AND+%22J+Exp+Med%22%5BJournal%5D)">The small GTPase Rap1b negatively regulates neutrophil chemotaxis and transcellular diapedesis by inhibiting Akt activation.</a> J Exp Med. 211, 1741-1758 (2014).</li>
</ol>

The authors have nothing to disclose.

This method enables TEM analysis on low cell numbers, by using Evans blue staining and agarose embedding to localize a tiny cell pellet during the dehydration, resin embedding and sectioning processes. Importantly, it maintains cell clusters together and preserves the cell ultra-structure, which is desirable to examine multiple cells for quantification of data.
In TEM studies, a cell pellet containing millions of cells is often required for efficient embedment into an agarose/gelatin matrix an...

Ultra-structural and sub-organelle details of cells have been mainly revealed by transmission electron microscopy (TEM) studies from tissues or cell pellets1,2. Solid pieces of tissues can be easily utilized for electron microscopy studies. However, TEM analyses1,3 on cells in suspension remain challenging and necessitate high cell numbers, i.e., millions of cells. Because of this, ultra-structural analyses of rare cell populations in suspension, e.g., hematopoietic stem cells (HSCs), are not easily assessed. Multiple attempts for TEM analysis from scarce samples using cytospin or monolayer approaches fail to get significant quantitative data from the limited number of cells. Thus, the requirement of a high cell number limits the use of this powerful tool to understand the subcellular ultrastructural details of rare cell populations.
A key limitation for TEM studies with a limited number of cells in suspension is the localization of the cells for processing and thus, TEM studies from limited cells with particularly small sizes remain challenging. Several alternative approaches have been adopted to counter this limitation: the BSA/bisacrylamide (BSA-BA) mediated polymerization of cell suspension, the staining of cells with a dye to make them visible on a thin film support including coverslips, and TEM analyses from cytospin preparations4-6. However, very limited success was achieved, as very few cells were found in the sections after ultra-sectioning. The key challenge of identifying sparse cells persists because the cell monolayer remains mostly invisible in the solidified gel for sectioning. Furthermore, cytospin preparation of cells may alter their cellular organization due to cell spreading and the fragility of the cell structure. Hence, these inherent drawbacks warrant a novel approach to perform TEM studies from rare cell populations with more consistency. To overcome this problem, we have described a novel and alternative sample preparation method for TEM studies from rare cell populations7.
Here, we report an efficient sample preparation protocol to perform TEM from scarce biological samples with consistent qualitative and quantitative results. Evans blue staining was performed after fixation to localize a tiny cell pellet from low number cells, i.e., 10,000 bone marrow hematopoietic stem and progenitor cells that would otherwise have remained invisible, and the pellet was embedded into agarose before dehydration and resin embedding processes. This method clusters the cells together and enables the efficient analysis of the ultrastructure and subcellular organization of hematopoietic stem cells (identified as Lin- Sca-1+ c-Kit+ Flt3- CD34-; HSC), a rare 0.2 - 0.5% cell population in the bone marrow. This experimental protocol can be useful to perform ultra-structural studies and obtain quantitative results on many rare but highly important populations.

<table><tbody><tr><td>Paraformaldehyde 20% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>15713</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Gluteraldehyde 70% Aqueous Soln</td><td>Electron microscopy Sciences</td><td>16350</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic via inhalation, skin contact. Wear gloves, use fume hood.</td></tr><tr><td>Cacodyladate buffer&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>12300</td><td>Make 0.1 M Cacodyladate buffer (pH 7.4) in distilled water, store at 4 &amp;deg;C. &lt;strong&gt;CAUTION&lt;/strong&gt; Irritant by inhalation or skin contact, wear gloves and work in fume hood.</td></tr><tr><td>Evans Blue&amp;nbsp;</td><td>Sigma</td><td>E-2129</td><td></td></tr><tr><td>Low melting agarose&amp;nbsp;</td><td>Sigma Aldrich</td><td>A-5030</td><td></td></tr><tr><td>Osmium tetraoxide</td><td>Electron microscopy Sciences</td><td>19130</td><td>1 g in 50 ml of 0.1 M cacodyladate buffer to make 2% OsO&lt;sub&gt;4&lt;/sub&gt; stock, &lt;strong&gt;CAUTION&lt;/strong&gt; Very toxic by inhalation or skin contact, strong oxidizer, wear protective clothing, eye protection, use fume hood.</td></tr><tr><td>&lt;strong&gt;LX-112 Embedding Kits (LADD&amp;reg;)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DDSA (Dodecenylsuccinic anhydride)</td><td>LADD</td><td>21340</td><td>Mix 16% DDSA; 30% NMA; 54% LX-112 together and then add 1.4% DMP-30</td></tr><tr><td>NMA (Nadic methyl anhydride)</td><td>LADD</td><td>21350</td><td></td></tr><tr><td>Epoxy Resins Epon 812 (LX-112)</td><td>LADD</td><td>21310</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, skin contact. Wear gloves, protective clothing, use fume hood.</td></tr><tr><td>DMP-30 (2,4,6-Tri(dimethylaminomethyl)phenol)</td><td>LADD</td><td>21370</td><td></td></tr><tr><td>Reynolds lead citrate (EM Stain II)</td><td>Leica</td><td></td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Uranyl acetate (EM Stain I)</td><td>Leica</td><td>8072820</td><td>&lt;strong&gt;CAUTION&lt;/strong&gt; Toxic by inhalation, avoid contact with skin or eyes.</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>ATCC</td><td>30-2005</td><td>composition: NaCl, NaHCO&lt;sub&gt;3&lt;/sub&gt;, HEPES, Glucose, Ca, Mg, sod pyruvate &lt;em&gt;etc.&lt;/em&gt; For details https://www.atcc.org/~/media/Attachments/E/9/7/E/4893.ashx</td></tr><tr><td>Pyramid tip mold&amp;nbsp;</td><td>Ted Pella</td><td>10585</td><td></td></tr><tr><td>mounting cylinders</td><td>Ted Pella</td><td>10580</td><td></td></tr><tr><td>Ultra-microtome&amp;nbsp;</td><td>(Leica EM UC7)</td><td></td><td></td></tr><tr><td>200 mesh grids (nikil)</td><td>Electron microscopy Sciences</td><td>G-200 Ni</td><td></td></tr><tr><td>Electron Microscope</td><td>Hitachi model H-7650&amp;nbsp;</td><td></td><td></td></tr><tr><td>Image capture engine software</td><td>AMT-600&amp;nbsp;</td><td></td><td></td></tr><tr><td>35 mm plastic Petri dishes</td><td>Fisher scientific</td><td></td><td></td></tr><tr><td>Quick Bond Super glue Cyanoacrylate&amp;nbsp;</td><td>Electron microscopy Sciences</td><td>72588</td><td></td></tr><tr><td>Razor blades</td><td></td><td></td><td></td></tr><tr><td>Glass scintillation vials&amp;nbsp;</td><td>Fisher scientific&amp;nbsp;</td><td>03-337-14</td><td></td></tr><tr><td>Glass slides&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Eyelash tool</td><td></td><td></td><td></td></tr><tr><td>Metal Loop tool&amp;nbsp;</td><td></td><td></td><td></td></tr></tbody></table>

an alternative approach for sample preparation with low cell number for tem analysis

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

An efficient and consistent protocol for sample preparation to perform TEM analysis from low number of cells is described. Post-fixation staining with Evans blue and the transfer of cells to a 0.5 ml microcentrifuge tube helped visualize the tiny cell pellet7. Osmification with osmium tetraoxide in agarose led to an easy detection of the dark cell pellet during the dehydration and embedding.
Semi-thin sections from th...

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

The overall goal of this procedure is to prepare samples with low cell numbers to perform TEM analysis in an efficient and reproducible manner. This method can help to reveal all structures of real cells and a better physiological settings in the biomedical field. The main advantage of this technique is that both qualitative and quantitative results can be obtained from low cell numbers in reproducible manner.To begin, use facts to sort 10, 000 hematopoietic stem cells, or HSC's into a 1.5 mL microcentrifuge tube. Containing 600 microliters of isocoves modified dulbecco medium with 10%FBS. Using a centrifuge with swing out buckets, spin down the sorted HSC's at 1, 000 times G for 10 minutes.Use gentile aspiration to remove all but 200 microliters of the medium. To fix the cells add 0.2 mL of freshly prepared two X fixative solution, and incubate at room temperature for one hour. Next add 600 microliters of 0.1 molar cacodylate buffer to wash the cells.Centrifuge the cells again, this time at 30 degrees Celsius. Then after centrifugation, and removing all but 200 microliters of buffer, add 200 microliters of two milligrams per milliliter of evans blue in cacodylate buffer, and incubate at room temperature for 20 minutes to stain the cells. Use 600 microliters of 0.1 molar cacodylate buffer to wash the cells three times.Then re-suspend the cells in the remaining 200 microliters of buffer before transferring the cell's suspension into a 0.5 mL tube. At this step the pellet should be visible in the tube. With a table top centrifuge, spin the cells at 1, 000 times G for 10 minutes.Then gently remove the buffer without disturbing the now visible tiny cell pellet, leaving 50 microliters of buffer in the tube. Prepare a 4%agarose solution, by adding 200 milligrams of low melting agarose to five milliliters of 0.1 molar cacodylate buffer in a 15 mL tube. Melt the agarose by transferring the tube to boiling water in a 100 mL glass bottle.Add 200 microliters of the agarose solution to the tube of the cell suspension. Immediately centrifuge at 1, 000 times G, and 30 degrees Celsius for 10 minutes. After confirming that the cells pelleted into the semisolid agarose at the bottom of the tube, immediately transfer the tube to four degrees Celsius, or ice, for 20 minutes to solidify the agarose.Using a 27 gauge needle, carefully transfer the solidified agarose, containing the cell pellet, to a 35 mL plastic petri dish containing 0.1 molar cacodylate buffer. With a scalpel trim the solidified agarose containing the cell pellet into one piece of about one to two millimeters. Then transfer it into a new 1.5 mL tube.Add 600 microliters of 0.1 molar cacodylate buffer and wash the agarose cell pellet by using a pipette to add and remove the buffer. Remove the buffer from the agarose cell pellet and in a fume hood, while wearing protective clothing and gloves, add one milliliter of 1%osmium tetroxide solution. Incubate the cell pellet at four degrees Celsius for one hour.After the incubation, wash the sample three times. Transfer it to a 20 mL glass scintillation vial with a cap. Next dehydrate the sample using an ethanol series.Then infiltrate the sample in increasing concentrations of LX 112 resin in ethanol before incubating in 100%resin two times for 60 minutes each time. Using forceps, carefully transfer the sample to the bottom of a pyramid tip shaped rubber mold, and add pure resin on top of the sample to fill the mold. Incubate the sample at 60 degrees Celsius for 72 hours to polymerize the resin.When the resin is polymerized, remove it from the molds by twisting the rubber mold. A small black cluster of cells should be visible in the polymerized resin pyramid. Use cyanoacrylate to attach the polymerized resin pyramid onto a mounting cylinder for sectioning.After mounting the block onto an ultramicrotome use a razor blade and diamond knife to trim the pyramid block, making a short, wide trapezoidal shape with the top and the bottom of the block parallel to each other, and with evenly angled sides. Then use the diamond knife to trim the block around the cell pellet and remove the plastic sections around the cell pellet agarose piece. With the ultramicrotome cut one micrometer sections and use an eyelash tool and metal loop to move the sections from the water boat to a glass slide.Then place the slides on a hot plate at 37 degrees Celsius to dry. Using a syringe with a 0.22 micrometer filter add a drop of teluatine blue solution to the sections and incubate for three minutes. Then with distilled water, rise the slides and let the sections dry.After drying check the sections under a light microscope to identify the position of the cells. Next use a diamond knife to cut ultra thin sections. Then with an eyelash tool, move two to three sections onto 200 mesh grids from the water boat and place in a glass petri dish.Transfer the dish with grids onto a hot plate at 30 degrees Celsius for 30 minutes to dry the sections. Add drops of 1%uranyl acetate, and incubate at room temperature for 10 minutes to stain the grids. Then, use distilled water to rinse six to eight times.Stain with Reynolds lead citrate at room temperature for five minutes before rinsing six to eight times. Transfer the glass petri dish containing the grids to a hot plate at 37 degrees Celsius to dry the sections. Finally, analyze the sections with an electron microscope at 87 kilovolts.Shown here at low magnification are semi-thin toluidine blue stained sections from blocks containing a tiny cell pellet that confirm a cluster of cells. This higher magnification image shows the intact morphology of the cells. This figure represents an over view field of the cell cluster from ultra thin sections that were stained with uranyl acetate and lead citrate and analyzed using electron microscopy.Shown here is a single HSC, demonstrating that the morphology and ultra structure of the cells were well preserved. A higher magnification highly detailed electron micrograph of an individual HSC shows the intracellular ultra structures. In addition, high magnification images captured well preserved structure and integrity of subcellular organelles such as mitochondria.Once mastered, this technique can be performed in five to six hours, and for advanced blue staining, and agarose embedding, and then normal TEM processing will take five to six days if it is performed properly. So after watching this video you sort of have a good understanding of how to prepare samples from real cell preparation for TEM Analysis.

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Miniaturized Sample Preparation for Transmission Electron Microscopy

This protocol describes the general processes and quality control checks necessary for preparing healthy adult mammalian single cells for droplet-based, high throughput single cell RNA-Seq preparations. Sequencing parameters, read alignment, and downstream single-cell bioinformatic analysis are also provided.

Droplet Barcoding-Based Single Cell Transcriptomics of Adult Mammalian Tissues

The present protocol describes a single-cell method for iterative epigenomic analyses using a reusable single cell. The reusable single cell allows analyses of multiple epigenetic marks in the same single cell and statistical validation of the results.

Reusable Single Cell for Iterative Epigenomic Analyses

The feasibility and effectiveness of high-throughput scRNA-seq methods herald a single-cell era in plant research. Presented here is a robust and complete procedure for isolating specific Arabidopsis thaliana root cell types and subsequent transcriptome library construction and analysis.

Isolation and Transcriptome Analysis of Plant Cell Types

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

This protocol outlines the procedure for rapidly dissociating human and mouse tumor samples for single-cell RNA sequencing.

Dissociation of Human and Mouse Tumor Tissue Samples for Single-cell RNA Sequencing

This protocol presents a practical method using Methanocaldococcus jannaschii (MjsHSP16.5) as an example to address uneven particle distribution through sample preparation optimization, providing a reference for researchers to efficiently elucidate macromolecular structures using cryogenic electron microscopy (cryo-EM).

Optimizing Sample Preparation for Cryogenic Electron Microscopy

Here, we present a protocol to automate single-cell histone post-translational modification analysis using an isotopic two-plex labeling strategy and ArgC digestion. This workflow enables quantitative, reproducible, and high-sensitivity profiling of chromatin heterogeneity and epigenetic responses at single-cell resolution.

Automated Sample Preparation for the Multiplexed Analysis of Single-Cell Histone Post-Translational Modifications (sc-hPTM2)

Learn an alternative TEM sample preparation method for low cell numbers using hematopoietic stem cells. This efficient protocol enables reproducible visualization of cellular ultrastructure in biomedical research.

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

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