This work was supported by National Institute of Health Grants R01HL089462 to SP.

1. Cationic Liposomes Preparation
<ol>
 <li>Autoclave a clean round-bottom flask to be used for lipososmes preparation.</li>
 <li>Prepare stock solutions:
 <ol class="lalpha">
 <li>- Dissolve 200 mg of dimethyl dioctadecyl ammonium bromide (DOAB) in 10 ml chloroform using a small glass bottle.</li>
 <li>- Dissolve 200 mg of cholesterol in 10 ml chloroform using a small glass bottle.</li>
 <li>- Prepare and autoclave to sterilize 5% glucose in 100 ml of RNA/DNA-ase free distilled water.</li>
 </ol>
 </li>
 <li>Rinse the round-bottom flask with chloroform. Add 5-10 ml to the flask, swirl, and let it sit for few minutes. Keep the flask covered with aluminum foil at all times. Empty the chloroform from the flask.</li>
 <li>Preparation of liposomes:
 <ol class="lalpha">
 <li>- Add 315 &mu;l of DOAB stock solution to the round-bottom flask.</li>
 <li>- Add 200 &mu;l of cholesterol stock solution to the flask.</li>
 <li>- Add 9.5 ml of chloroform to the flask and mix by swirl.</li>
 </ol>
 </li>
 <li>Set the rotavapor at 37 &deg;C, 100 rotations per minute (rpm) or more (100-120 rpm). The rotavapor should be connected to a water-pump vacuum control with a suction tube properly attached.</li>
 <li>Connect the argon gas tank and the rotavapor.</li>
 <li>Attach the flask to the rotavapor and adjust the optimal settings before lowering it in the water bath. Open the first valve for the argon completely by turning it counterclockwise. Always open and close this valve first. The second valve controls the argon pressure blowing in to the flask. The pressure should be always gentle.</li>
 <li>Lower the rotavapor in the water bath, enough for the flask to touch the water without being completely submerged and turn on the speed.</li>
 <li>Wait until all the chloroform evaporates, about 10 min. Then stop the machine.</li>
 <li>Turn off the rotavapor speed and the argon tank valves. Now the flask should be removed.</li>
 <li>Add 2 ml of 5% glucose to the flask to dissolve the lipids.</li>
 <li>Scratch the bottom of the flask thoroughly using 1 ml pipette tip to dissolve all lipids. After scratching, remove any remaining lipid from the 1 ml tip into the flask, if necessary, with a second tip.</li>
 <li>Incubate the obtained solution in 42 &deg;C water bath while slowly vortexing (15 rpm) the flask.</li>
 <li>Fill the sonicator vat with cold water. Sonicate the solution for at least 20-30 min with the flask held up and the bottom just barely touching the water.</li>
 <li>Heat the flask in the rotavapor's water bath for 20 min at 42 &deg;C, at zero speed, with the round bottom immersed in water, this time.</li>
 <li>Filter the liposomes through a 0.47 micron and subsequently, 0.22 micron syringe filters.</li>
 <li>Using a small extruder, filter the liposomes through a 50 nm membrane to generate a homogenous population of unilamellar liposome vesicles.</li>
 <li>Transfer the liposomes to an Eppendorf tube and place it on ice.</li>
</ol>
2. Prepare the Liposomes: siRNA-ITSN-1 Complexes
<ol>
 <li>Resuspend the on-target mouse siRNAITSN in siRNA buffer containing 20 mM HEPES and 150mM sodium chloride, pH 7.4, to a final concentration of 2 &mu;g/&mu;l. Keep it on ice before mixing.</li>
 <li>Prepare the liposomes:siRNAITSN complexes at a ratio of 8 nmol liposomes:2 &mu;g RNA (or 400 nmol liposomes:100 &mu;g siRNAITSN). In the present study, we added 50 &mu;l of siRNAITSN from the stock solution (50 &mu;M) to 100 &mu;l of freshly prepared liposomes using a RNA/DNA-ase free Eppendorf tube. Note that the concentration of siRNAITSN is very important because a maximum of 150 &mu;l of mixture can be bilaterally and retro-orbital injected in the mouse at one time.</li>
 <li>Keep the mixture on ice while waiting to inject the mice.</li>
</ol>
3. Mouse Retro-orbital Vein Injection (Internal Angle of the Eye Socket)
All mouse experiments were approved and performed in accordance with the guidelines of Rush University Institutional Animal Care and Use Committee.
<ol>
 <li>Anesthetize the mouse by intra-peritoneal injection of a 50 mg/kg body weight ketamine and 5 mg/kg body weight xylazine (commercially available mixture). Depending on the animal weight and age, adjust the anesthetics volume and concentration. The mice used in the study mice are CD1 male mice, 6-8 weeks old and weigh around 25 grams. The syringes are tuberculin 1 ml type with the needle attached.</li>
 <li>Hold mouse ears with one hand and turn the mouse down on a side to expose the internal angle of the eye. Aim medial to the plica semilunaris; avoid touching the eyeball.</li>
 <li>Approach the lacrimal caruncle of the eye holding the syringe containing the mixture at a 45 degrees angle in all three axes.</li>
 <li>Slowly inject the mixture and let the mouse to recover with the injected side up. If a large liquid droplet forms at the needle insertion place, retract the needle, suction back the droplet into the syringe and try again. Repeated injections are best done by alternating the eyes, to allow perfect recovery. If unsure, this technique can be checked by injecting blue dye (50 &mu;l, 0.5% crystal violet in methanol) and exposing the mouse lungs to observe the blue vasculature.</li>
 <li>The mice in this study were injected every 3rd day for 3 weeks without fatalities or adverse effects.</li>
</ol>
4. Mouse Lung Perfusion and Tissue Collection for Biochemical Studies
<ol start="0">
 <li>Set the peristaltic pump to run at 1.5 ml/min.</li>
 <li>Prepare the setting: ventilator, operating table, instruments, sterile cotton swabs, q-tips, strings, beakers, distilled water, Hank's balanced salt, tracer.</li>
 <li>Anesthetize the mouse by intraperitoneal injection of the anesthetic mixture (100 mg/kg body weight ketamine and 10 mg/kg body weight xylazine).</li>
 <li>At the neck level, remove the salivary glands and expose the trachea.</li>
 <li>Start with laparotomy, excise the diaphragm, and follow with thoracotomy, exposing the lungs and the heart.</li>
 <li>Remove the thymus.</li>
 <li>Using the light microscopy for better accuracy, catheterize the pulmonary artery.</li>
 <li>Do tracheostomy and intubate the mouse using the tracheostoma. Set the ventilator to a rate of 150 strokes/min and stroke volume of 150 &mu;l.</li>
 <li>As outlet, open the left atrium.</li>
 <li>Perfuse the mouse lung vasculature free of blood for 5 min, using the peristaltic pump and Hank's solution warmed at 37 &deg;C.</li>
 <li>Perfuse the tracer (8 nm gold-albumin16) for additional 10 min at the same flow rate.</li>
 <li>Flush the unbound tracer by 5 min lung perfusion with Hank's solution.</li>
 <li>Follow with in situ fixation of the lungs by 10 min perfusion of 4% (wt/vol)formaldehyde, 2.5% glutaraldehyde, and 1% tannic acid in 0.1 PIPES buffer, pH 7.2.</li>
 <li>Collect the lungs, remove the excessive tissue, cut small blocks (3/3 mm), and place them in to a labeled scintillation vial containing 2 ml of fixative mixture. </li>
</ol>
 
The mice will expire fully anesthetized during the procedure.
5. Mouse Lung Tissue Processing for EM
<ol>
 <li>Prepare stock solutions: 1% Palade OsO4, acetate veronal, and Kellenberger buffer.
 <ol class="lalpha">
 <li>- 1% Palade-OsO4 buffer contains: 1 ml acetate veronal stock, 1.25 ml 4% OsO4, 1 ml of 0.1 N hydrochloric acid, and distilled water up to 5 ml of final volume.</li>
 <li>- Acetate veronal stock solution is obtained by dissolving 1.15 g sodium acetate andydrous and 2.94 g barbital in 100 ml of distilled water.</li>
 <li>-Kellenberger solution contains 2 ml of acetate veronal stock, 2.8 ml of 0.1 N hydrochloric acid, 0.05 g uranyl acetate, and 5.1 ml of distilled water, pH= 6.</li>
 </ol>
 </li>
 <li>Wash the lung blocks in 0.1 M sodium cacodylate buffer, pH= 7.4, briefly 3 times.</li>
 <li>Fix the specimens in the 4% (wt/vol) formaldehyde, 2.5% glutaraldehyde in 0.1 PIPES buffer, pH 7.2, for 1 hr, at room temperature.</li>
 <li>Post-fix with 1% Palade-Osmium for 1 hr in the hood, on ice, in the dark.</li>
 <li>Rinse once with Kellenberger buffer.</li>
 <li>Incubate specimens in Kellenberger buffer for 2 hr to overnight, at room temperature.</li>
 <li>Rinse once in 50% ethanol.</li>
 <li>Dehydrate with graded series of ethanol, 5 min/each: 70%, 95%, then 2 x 15 min 100% ethanol.</li>
 <li>Switch to 100% propylene oxide, 2 x 15 min.</li>
 <li>Remove the propylene oxide and add a mixture of 50% propylene oxide and 50% Epon 812, incubate overnight, rotating on a wheel, at room temperature.</li>
 <li>Next morning, remove the mixture and add fresh Epon 812, at least for 4-5 hr on the wheel.</li>
 <li>Using doubled tapered molds, filled half way with fresh 100% Epon 812, add selected specimens and place them to the edges.</li>
 <li>Move the molds to the incubator set at 60 &deg;C and let them cure for 48-72 hr.</li>
 <li>When polymerized, send the blocks to be thin (60-70 nm thick) sectioned.</li>
</ol>

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Chem. 282 (282), 17166-17178 (2007).</li><li>Uddin, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cationic+lipids+used+in+non-viral+gene+delivery+systems.">Cationic lipids used in non-viral gene delivery systems.</a> Biotechnology and Molecular Biology Review. 2 (2), 58-67 (2007).</li><li>Barenholz, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+lipid+composition+on+the+thermotropic+behavior+and+size+distribution+of+mixed+cationic+liposomes.">Influence of lipid composition on the thermotropic behavior and size distribution of mixed cationic liposomes.</a> J. Colloid Interface Sci. (356), 46-53 (2011).</li><li>Predescu, D. N., Neamu, R., Bardita, C., Wang, M., Predescu, S. 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The authors have nothing to disclose.

Based on previous studies published by others9 and us1, 18 we developed this methodology for long term knock down of ITSN-1s in vivo by repeated intravenous administration (every 72 hr, for 24 consecutive days) of specific siRNA/liposome complexes. This experimental approach is efficient, can be used safely and repeatedly and it can be easily extended to study the involvement of a gene encoding any protein of interest in lung endothelium and pulmonary homeostasis. Under the experimen...

Naked siRNA cannot penetrate the cell membrane, being negatively charged, and it is easily degraded by enzymes in blood, tissues, and cells. Even with recently structural modifications to improve stability, siRNA accumulation at the target site after administration is extremely low and requires an efficient intracellular vehicle5. Cationic liposomes emerged as safe nucleic acids carriers with the potential to transfer large pieces of DNA/RNA into cells by encapsulating and protecting the nucleic acids from enzymatic degradation6. Also, cationic liposomes spontaneously interact with DNA/RNA, thus promoting gene transfer to the cells2. More recently liposomes have been applied to deliver vaccines and low-molecular-weight drugs7. Liposomal delivery of microRNA-7-expressing plasmid overcomes epidermal growth factor receptor tyrosine kinase inhibitor-resistance in lung cancer cells8. When targeting the vascular endothelium, the intravenous delivery is essential because the complexes are unlikely to cross the endothelial barrier and extravasate in to the interstitium9. By comparison to other organs, ECs from the microvasculature of the lung have the greatest uptake and avidly internalize cationic liposome and DNA/RNA complexes, followed by the lymph nodes and Peyer's patches9. Intravenous delivery in rodent models of siRNA via retro-orbital or tail vein injections has been proven harmless even in high concentrations, such as 50 mg/kg10. In the published literature the composition of cationic liposomes differs, based on the lipids formulation and their equimolar ratio11, 12. There is a wide array of potential applications using cationic liposomes for gene delivery in vivo, whether targeting down-regulation/over-expression of the proteins, delivery of vaccines or anti-tumor therapies13-15. Important to remember is that the efficiency of DNA/RNA-cellular membrane interaction is regulated by the shape coupling between generated complexes and membrane lipids. This suggests that tailoring the lipids composition to the targeted cellular membrane profiles may be beneficial and result in high rates of transfection in a particular cell type6.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethyl Dioctadecyl Ammonium Bromide (DOAB)</td>
 <td>Sigma-Aldrich</td>
 <td>D2779</td>
 <td></td>
 </tr>
 <tr>
 <td>LiposoFast stabilizer (mini-extruder)</td>
 <td>Avestin Inc.</td>
 <td>LF-STB</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate membrane, 19 mm diameter, 50 nm pore</td>
 <td>Avestin Inc.</td>
 <td>LFM-50</td>
 <td></td>
 </tr>
 <tr>
 <td>Cholesterol</td>
 <td>Sigma-Aldrich</td>
 <td>C-8667</td>
 <td></td>
 </tr>
 <tr>
 <td>Chloroform</td>
 <td>Mallinckrodt Chemicals</td>
 <td>4432-04</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's balanced salts</td>
 <td>Sigma-Aldrich</td>
 <td>H1387</td>
 <td></td>
 </tr>
 <tr>
 <td>Round-bottom flask</td>
 <td>Fisher Scientific</td>
 <td>FHB-275-030X</td>
 <td></td>
 </tr>
 <tr>
 <td>Mouse siRNAITSN - on target </td>
 <td>Dharmacon/ThermoScientific</td>
 <td>J-046912-11</td>
 <td></td>
 </tr>
 <tr>
 <td>Peristaltic pump</td>
 <td>Ismatec</td>
 <td>REGLO-CDF digital with RHOO pump head</td>
 <td></td>
 </tr>
 <tr>
 <td>Ventilator</td>
 <td>Hugo Sachs Electronik</td>
 <td>NA</td>
 <td></td>
 </tr>
 <tr>
 <td>Barbital</td>
 <td>Sigma-Aldrich</td>
 <td>B-0500</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>EM Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>Epon812</td>
 <td>EM Sciences</td>
 <td>Discontinued by the manufacturer</td>
 <td></td>
 </tr>
 <tr>
 <td>Propylene oxide</td>
 <td>EM Sciences</td>
 <td>20400</td>
 <td></td>
 </tr>
 <tr>
 <td>Embedding molds</td>
 <td>EM Sciences</td>
 <td>69923-05</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>EM Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 <tr>
 <td>8 nm gold-albumin tracer</td>
 <td></td>
 <td>Prepared in the laboratory as in16</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Comments</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipped with 2 Avestin 1 ml gas-tight syringes (cat. # LF-1)</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Pyrex, 100 ml</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Modified siRNA, in vivo</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Part of IDEX corp.</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>D-79232 March, Germany</td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Replaced with Embed812, cat. # 14120</td>
 </tr>
 </tbody>
</table>

long-term silencing of intersectin-1s in mouse lungs by repeated delivery of a specific sirna via cationic liposomes. evaluation of knockdown effects by electron microscopy

Previous studies showed that knockdown of ITSN-1s (KDITSN), an endocytic protein involved in regulating lung vascular permeability and endothelial cells (ECs) survival, induced apoptotic cell death, a major obstacle in developing a cell culture system with prolonged ITSN-1s inhibition1. Using cationic liposomes as carriers, we explored the silencing of ITSN-1s gene in mouse lungs by systemic administration of siRNA targeting ITSN-1 gene (siRNAITSN). Cationic liposomes offer several advantages for siRNA delivery: safe with repeated dosing, nonimmunogenic, nontoxic, and easy to produce2. Liposomes performance and biological activity depend on their size, charge, lipid composition, stability, dose and route of administration3Here, efficient and specific KDITSN in mouse lungs has been obtained using a cholesterol and dimethyl dioctadecyl ammonium bromide combination. Intravenous delivery of siRNAITSN/cationic liposome complexes transiently knocked down ITSN-1s protein and mRNA in mouse lungs at day 3, which recovered after additional 3 days. Taking advantage of the cationic liposomes as a repeatable safe carrier, the study extended for 24 days. Thus, retro-orbital treatment with freshly generated complexes was administered every 3rd day, inducing sustained KDITSN throughout the study4. Mouse tissues collected at several time points post-siRNAITSN were subjected to electron microscopy (EM) analyses to evaluate the effects of chronic KDITSN, in lung endothelium. High-resolution EM imaging allowed us to evaluate the morphological changes caused by KDITSN in the lung vascular bed (i.e. disruption of the endothelial barrier, decreased number of caveolae and upregulation of alternative transport pathways), characteristics non-detectable by light microscopy. Overall these findings established an important role of ITSN-1s in the ECs function and lung homeostasis, while illustrating the effectiveness of siRNA-liposomes delivery in vivo.

ITSN-1s protein and mRNA levels were monitored at several time points after siRNAITSN delivery by Western blot and conventional and quantitative PCR as in4. ITSN-1s protein and mRNA levels in siRNAITSN-treated mouse lungs were about 75% lower by reference to controls during continuous knockdown of ITSN-1s for 21 days. Without ITSN-1s, dynamin-2, a major interacting partner of ITSN-1s and essential player in detachment of caveolae from the plasma membrane is not efficiently recruited to th...

Watch this Scientific Journal Video about Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Elect

Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Previous  studies showed that knockdown of ITSN-1s (KDITSN), an endocytic  protein involved in regulating lung vascular permeability and endothelial cells ...

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10.6K Views.  Rush University. Repeated retro-orbital injections of cationic liposomes/siRNA/ITSN-1s complexes in mice, every 72 hours for 24 days, efficiently deliver the siRNA duplex to the mouse lung microvasculature reducing ITSN-1s mRNA and protein expression by 75%. This technique is highly reproducible in an animal model, has no adverse effects and avoids fatalities.

Video: Long-term Silencing of Intersectin-1s in Mouse Lungs by Repeated Delivery of a Specific siRNA via Cationic Liposomes. Evaluation of Knockdown Effects by Electron Microscopy

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

Długoterminowe Imaging komórkach ssaków przy dużym polu Mikroskopia

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Cryo-electron Microscopy Specimen Preparation By Means Of a Focused Ion Beam

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms or solutions. We make use of a custom built cryo-transfer station and a modified cryo-SEM preparation chamber2. The spores are taken from a culture, plunge-frozen in a liquid nitrogen slush and observed in the cryo-SEM to select a region of interest. A thin lamella is then extracted using the FIB, attached to a TEM grid and subsequently thinned to electron transparency. The grid is transferred to a cryo-TEM holder and into a TEM for high resolution studies. Thanks to the introduction of a cooled nanomanipulator tip and a cryo-transfer station, this protocol is a straightforward adaptation to cryogenic temperature of the routinely used FIB preparation of TEM samples. As such it has the advantages of requiring a small amount of modifications to existing instruments, setups and procedures; it is easy to implement; it has a broad range of applications, in principle the same as for cryo-TEM sample preparation. One limitation is that it requires skillful handling of the specimens at critical steps to avoid or minimize contaminations.

Cryo Electron Microscopes, either Scanning (SEM) or Transmission (TEM), are widely used for characterization of biological samples or other materials with a high water content1. A SEM/Focused Ion Beam (FIB) is used to identify features of interest in samples and extract a thin, electron-transparent lamella for transfer to a cryo-TEM.

Here we present a protocol used to prepare cryo-TEM samples of Aspergillus niger spores, but which can easily be adapted for any number of microorganisms ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Correlative Confocal and 3D Electron Microscopy of a Specific Sensory Cell

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused throughout tissues made of diverse cell types, such as enteroendocrine cells of the intestinal epithelium. These gastrointestinal sensors of food and bacteria have been difficult to study because they are dispersed among other epithelial cells at a ratio of 1:1,000. Recently, transgenic reporter mice have been generated to identify enteroendocrine cells by means of fluorescence. One of those is the peptide YY-GFP mouse. Using this mouse, we developed a method to correlate confocal and serial block-face scanning electron microscopy. We named the method cocem3D and applied it to identify a specific enteroendocrine cell in tissue and unveil the cell&#8217;s ultrastructure in 3D. The resolution of cocem3D is sufficient to identify organelles as small as secretory vesicles and to distinguish cell membranes for volume rendering. Cocem3D can be easily adapted to study the 3D ultrastructure of other specific cell types in their native tissue.

Here, we introduce a method, cocem3D, to unveil the ultrastructure of a specific cell in its native tissue by bridging confocal and serial block-face scanning electron microscopy.

Delineation of a cell&#8217;s ultrastructure is important for understanding its function. This can be a daunting project for rare cell types diffused ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Meiotic Spindle Assessment in Mouse Oocytes by siRNA-mediated Silencing

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon fertilization. The resulting aneuploidy in developing embryos is recognized as a major cause of pregnancy loss and congenital birth defects such as Down&#8217;s syndrome. Accurate chromosome segregation is critically dependent on the formation of the microtubule spindle apparatus, yet this process remains poorly understood in mammalian oocytes. Intriguingly, meiotic spindle assembly differs from mitosis and is regulated, at least in part, by unique microtubule organizing centers (MTOCs). Assessment of MTOC-associated proteins can provide valuable insight into the regulatory mechanisms that govern meiotic spindle formation and organization. Here, we describe methods to isolate mouse oocytes and deplete MTOC-associated proteins using a siRNA-mediated approach to test function. In addition, we describe oocyte fixation and immunofluorescence analysis conditions to evaluate meiotic spindle formation and organization.

Here, we present a protocol for specific siRNA-mediated mRNA depletion followed by immunofluorescence analysis to evaluate meiotic spindle assembly and organization in mouse oocytes. This protocol is suitable for in vitro depletion of transcripts and functional assessment of different spindle and/or MTOC-associated factors in oocytes.

Errors in chromosome segregation during meiotic division in gametes can lead to aneuploidy that is subsequently transmitted to the embryo upon ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...