This work was supported by NSF HRD-0630456, an NSF CREST Program and NSF is DMR-0934142.

1. Synthesis of &alpha;,&omega;-dihydroxyl Polymer [HO-PEO-P2MS-PEO-OH]
<ol>
 <li>Assemble polymerization reactor as shown in Figure 1. The reactor for this experiment consist of a 100 ml round bottom 2-neck flask having a standard taper outer joint (Chemglass), two flow control adapters with stopcocks (Chemglass), and one Teflon stirring rod. Adapter A (Figure 1) was used to keep Ultra High Purity(UHP) Nitrogen flowing through the system in order to prevent air and moisture from entering the inert system. Adapter B (Figure 1) was used to inject the solvent, monomer, and initiator into the reaction flask.</li>
 <li>Dry 200 ml of Tetrahydrofuran (THF) over Na metal, using benzophenone as indicator, for a minimum of 6 hr under dry nitrogen gas.</li>
 <li>Dry 10 ml of 2-methoxystyrene over calcium hydride for 24 hr.</li>
 <li>Prepare a cold temperature bath maintained at -78 &deg;C using a slurry of isopropanol and liquid nitrogen.</li>
 <li>Add 25 ml of THF into the polymerization reaction flask (see Figure 1) under nitrogen gas and keep reactor under nitrogen gas all through the polymerization.</li>
 <li>Place 100 ml round bottom flask into slurry.</li>
 <li>Add 2 ml (0.27 mmol/ ml) of the initiator solution into the reaction flask.</li>
 <li>Inject the first monomer, 2-methoxystyrene (4 ml) into the reaction flask.</li>
 <li>Allow the reaction to proceed for 40 min.</li>
 <li>Add 1 ml of the second monomer, ethylene oxide.</li>
 <li>Allow the polymerization to continue at room temperature for two days.</li>
 <li>Terminate the polymer with HCl (6 M)/methanol (1/20, vol/vol).</li>
 <li>Purify the polymer by precipitating into hexanes and dry polymer in a vacuum oven.</li>
 <li>Characterize the polymer using NMR.</li>
</ol>
2. Functionalization of &alpha;,&omega;-dihydroxyl Polymer with N-2,4-DNP-&Epsilon;-amino Caproic Acid to Obtain the Functional Polymer, CDNP-PEO-P2MS-PEO-CDNP
<ol>
 <li>In a three neck flask, place the &alpha;,&omega;-dihydroxyl polymer (0.05 mmol), the N-2,4-DNP-E-amino caproic acid (0.25 mmol), DCC (0.15 mmol) and DMAP (0.005 mmol) and dry on vacuum line for 4 hr.</li>
 <li>Distill dry dichloromethane (10 ml) into the flask.</li>
 <li>Release the vacuum under nitrogen and stir reaction for 12 hr at room temperature.</li>
 <li>Filter reaction mixture and recover polymer by precipitating twice into hexanes and methanol.</li>
 <li>Dry precipitated polymer in a vacuum oven at 40 &deg;C.</li>
 <li>Determine the polymer structure and functionality by FT-IR and 1H NMR.</li>
</ol>
3. Preparation of CDNP-PEO-P2MS-PEO-CDNP/SWCNT Solution for Electrospinning
<ol>
 <li>Dissolve 20 w % of CDNP-PEO-P2MS-PEO-CDNP in chlorobenzene.</li>
 <li>Dissolve 20 w % and 40 w % of Polystyrene (MW 800,000) in chlorobenzene to prepare two solutions. The higher molecular polystyrene is used to increase polymer chain-chain entanglement and obtain the optimum viscosity required for electrospinning.</li>
 <li>Mix the polymer solutions prepared in 3.1 and 3.2 together to form 1:1 and 1:2 ratios of the polymers and add 1 w % Single walled carbon nanotubes (SWCNT) to the mixture and stir overnight for even distribution of CNTs.</li>
</ol>
4. Electrospinning of Polymer-CNT Composite
<ol>
 <li>Assemble the electrospinning set up as shown in Figure 2. On the right hand side of the figure is the Glassman High Voltage source. Next to it is a retort stand on which the silicon wafer is attached. To the left is another retort stand on which the syringe is mounted and behind it is the lamp for visualizing the procedure as it progresses.</li>
 <li>Using a hypodermic syringe, withdraw a small quantity of the CDNP-PEO-P2MS-PEO-CDNP/polystyrene/SWCNT mixture (about 1 ml) and mount the hypodermic syringe on the retort stand.</li>
 <li>A silicon wafer is mounted across from the syringe securely at a distance of 10 cm, and the ground clip of the high voltage source is attached to it.</li>
 <li>Attach the clip bearing the high voltage to be applied to the needle on the syringe, depress the plunger a little bit (to suspend a drop on the needle tip)and at this point, electrospinning is ready.</li>
 <li>Power on the high voltage source and adjust voltage meter to 10 kV. Depending on the nature of the polymers in the composite, higher voltages may be required, especially if nanofibers under a hundred nanometers in diameter are desired.</li>
 <li>Unmount silicon wafer and place in a dessicator overnight to dry completely.</li>
</ol>
5. Characterization of Nanofibers
<ol>
 <li>Initial imaging of nanofibers is done with optical microscope to observe the overall perspective of the fibers.</li>
 <li>Utilize Scanning Electron Microscope to observe finer details like morphology, diameter, average pore size, etc.</li>
 <li>Carry out further imaging with an Atomic Force Microscope to observe 3-D topography of fibers, etc.</li>
</ol>
6. Binding Specificity of Nanofibers with Anti-DNP IgE Protein
<ol>
 <li>Prepare a solution of 4 ug/l fluorescently labeled, FITC- IgE (Fluorescein Isothio- cyanate-Immunoglobulin E) in PBS-BSA (Phosphate Buffered Saline-Bovine Serum Albumin) solution.</li>
 <li>Place a small piece of silicon wafer on which there are nanofibers on a MatTek well coverslip. Incubate the nanofibers in this solution for one hr. Incubation is done by gently pipetting out, 10 ul IgE solution onto the silicon wafer.</li>
 <li>After incubation, remove unbound IgE by washing sample three times with the PBS-BSA buffer solution. The PBS solution is dispensed gently on the wall of the MatTek dish, in order to avoid squirting the buffer directly onto the nanofibers. Swirl the dish gently by hand, to distribute buffer solution on nanofibers. Carefully remove buffer with a pipette and repeat this two more times.</li>
 <li>For the control, incubate nanofibers in fluorescently labeled IgG (non-specific for DNP) under the same conditions.</li>
 <li>Visualize the bound fibers with a Confocal Microscope to observe binding with IgE. For our study, the microscope used was Leica confocal TCS SP2 with 63x lens.</li>
</ol>
7. Current-Voltage Behavior of Nanofibers
 
<ol>
 <li class="jove_content">Connect two micro positioners to a very low current source like the Keithley 6430 sensitive sourcemeter. The set up for determining the current voltage behavior is shown in Figure 3. This figure shows the Probe station used to determine the initial I-V characteristics of the nanofibers. It is made up of the Bausch and Lomb MicroZoom Microscope, a Vacuum Chuck Stage, and four Micropositioners used in probing. On the top right is the Agilent 34405A Digital Multimeter used in measuring the voltage and below that is the Keithley 6430 Sub-Femtoamp Remote Source Meter used to source the low currents that were input into the fibers.</li>
 <li class="jove_content">Mount the probe arms of the micro positioners over the fiber mat on opposite sides with the tips touching the fibers.</li>
 <li class="jove_content">Connect another two micro positioners to a Digital multimeter, mount the probe arms in-between the other two and land the tips on the fiber mat. Ensure that the four tips are as collinear as possible.</li>
 <li class="jove_content">Input varying amounts of current from the Keithley (typically in the nanoamps range).</li>
 <li class="jove_content">Measure the voltage drop across the outer tips, for every magnitude of current sourced.</li>
 <li class="jove_content">Plotting these values will indicate the type of device the fiber mat acts like.</li>
</ol>
 
8. Representative Results
Functional Polymer
The method for the synthesis of &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP) is shown in Figure 4.1 The structure of the functional polymer was confirmed by FT-IR (Figure 5) and 500 MHz 1H NMR spectroscopy (Figure 6). The FT-IR shows the complete disappearance of the -OH broad absorption around 3,500 cm-1 indicating quantitative functionalization with the CDNP group. This is also confirmed by the NMR spectrum shown in Figure 6. Using the integration of the peaks in the NMR spectrum, it was determined that the CDNP-PEO-P2MS-PEO-CDNP polymers are quantitatively functionalized.
Nanofibers
In Figure 7, a mat of conductive nanofibers obtained by electrospinning CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT from chlorobenzene is shown. Confocal images obtained showed that the protein IgE binds with the DNP on the fiber surface.3 This is an indication of the specificity of binding of electrospun DNP-polymers towards IgE antibody. The intensity of light is an indicator of the presence of IgE on the nanofibers as the protein is fluorescently tagged.
Figure 8a is an AFM (Atomic Force Microscope) image of one the nanofibers obtained by this process and Figure 8b shows the dimension of this particular nanofiber is around 150 nm in diameter. By this process fibers between 100-700 nm are obtained. At this current time it is challenging to prepare fibers with a specific dimension. This is consistent with what is observed by other groups.4 Figure 9 shows SEM images of CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT nanofibers and the diameter of the nanofibers were between 200 nm to 300 nm .There are three SEM images of nanofibers shown at different magnifications. Study of the three images shows the morphologies of the fibers are linear and beaded. The overall goal is to prepare fibers which are mostly linear. Figure 10 shows the I-V plot of mats of nanofibers prepared from CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT. The plot shows behavior of a resistor (Ohmic). When the antigen is bound to the nanofibers, we expect to see a change In the I-V behavior of the fiber mat as this change in resistance is a characteristic which suggests that the functional fibers have potential application as the active component in sensors for single molecule detection.
<img alt="Figure 1" src="/files/ftp_upload/4135/4135fig1.jpg" /> 
Figure 1. Polymerization reactor for synthesizing the &alpha;,&omega;-dihydroxyl polymer. A) The injection point for the flow UHP gas nitrogen. B.) Injection point for the solvent, monomer, and initiator. C) The reaction vessel.
<img alt="Figure 2" src="/files/ftp_upload/4135/4135fig2.jpg" /> 
Figure 2. Setup used for electrospinning using a Glassman high voltage source.
<img alt="Figure 3" src="/files/ftp_upload/4135/4135fig3.jpg" /> 
Figure 3. Setup used to measure I-V plots using a Sub-femtoamp Remote Sourcemeter (Keithley).
<img alt="Figure 4" src="/files/ftp_upload/4135/4135fig4.jpg" /> 
Figure 4. A).Synthetic approach for preparing OH-PEO-P2MS-PEO-OH polymers. B) Functionalization of &alpha;,&omega;-dihydroxy[poly(ethyleneoxide)-b-poly( 2-methoxystyrene)-b-poly(ethyleneoxide)].
<img alt="Figure 5" src="/files/ftp_upload/4135/4135fig5.jpg" /> 
Figure 5. FT-IR spectra of (A) OH-PEO-P2MS-PEO-OH, precursor to CDNP-PEO-P2MS-PEO-CDNP and (B) CDNP-PEO-P2MS-PEO-CDNP.
<img alt="Figure 6" src="/files/ftp_upload/4135/4135fig6.jpg" /> 
Figure 6. 500 MHz Proton NMR of CDNP-PEO-P2MS-PEO-CDNP.
<img alt="Figure 7" src="/files/ftp_upload/4135/4135fig7.jpg" /> 
Figure 7. A) Binding image of FITC- IgE with CDNP-PEO-P2MS-PEO-CDNP fibers electrospun from chlorobenzene. B) Confocal microscope image of the control (nanofibers with IgG).
<img alt="Figure 8" src="/files/ftp_upload/4135/4135fig8.jpg" /> 
Figure 8. A) AFM image of CDNP-PEO-P2MS-PEO-CDNP Fibers electrospun from chlorobenzene and B) AFM profile i.e. dimension of one fiber shown in Figure 5a.
<img alt="Figure 9" src="/files/ftp_upload/4135/4135fig9.jpg" /> 
Figure 9. SEM images of CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT nanofibers.
<img alt="Figure 10" src="/files/ftp_upload/4135/4135fig10.jpg" /> 
Figure 10. I-V plot of mats of nanofibers prepared from CDNP-PEO-P2MS-PEO-CDNP /polystyrene/SWCNT.

<ol>
	<li>Sannigrahi, B., Sil, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+Characterization+of+&#945;,&#969;-bi[2,4-dinitrophenyl+(DNP)]+poly(2-methoxystyrene)+Functional+Polymers.+Preliminary+Evaluation+of+the+Interaction+of+the+Functional+Polymers+with+RBL+Mast+Cells.">Synthesis and Characterization of &#945;,&#969;-bi[2,4-dinitrophenyl (DNP)] poly(2-methoxystyrene) Functional Polymers. Preliminary Evaluation of the Interaction of the Functional Polymers with RBL Mast Cells.</a> Journal of Macromolecular Science, Part A. 45, 664-671 (2008).</li><li>Gordon, K., Sannigrahi, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+Optically+Active+Helical+Poly(2-methoxystyrene).+Enhancement+of+HeLa+and+Osteoblast+Cell+Growth+on+Optically+Active+Helical+Poly(2-methoxystyrene)+Surfaces.">Synthesis of Optically Active Helical Poly(2-methoxystyrene). Enhancement of HeLa and Osteoblast Cell Growth on Optically Active Helical Poly(2-methoxystyrene) Surfaces.</a> Journal of Biomaterials Science. 2, 2055-2072 (2009).</li><li>Baird, E. J., Holowka, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+Effective+Poly(Ethylene+Glycol)+Architectures+for+Specific+Inhibition+of+Immune+Receptor+Activation.">Highly Effective Poly(Ethylene Glycol) Architectures for Specific Inhibition of Immune Receptor Activation.</a> Biochemistry. 2, 12739-12748 (2003).</li><li>Ramakrisna, S., Fugihara, K., Lim, W. -. E., Ma, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Introductions to Electrospinning and Nanofibers. , (2005).</li><li>Kameoka, J., Craighead, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fabrication+of+Oriented+Polymeric+Nanofibers+on+Planar+Surfaces+by+Electrospinning.">Fabrication of Oriented Polymeric Nanofibers on Planar Surfaces by Electrospinning.</a> Applied Physics Letters. 83, 371-3773 (2003).</li><li>Ramakrishna, S., Lala, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymer+Nanofibers+for+Biosensor+Applications.">Polymer Nanofibers for Biosensor Applications.</a> Topics in Applied Physics. 109, 377-392 (2007).</li><li>Reuven, D., Sil, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Archetypical+Conductive+Polymer+Structure+for+Specific+Interaction+with+Proteins.">Archetypical Conductive Polymer Structure for Specific Interaction with Proteins.</a> Journal of Macromolecular Science Part A: Pure and Applied Chemistry. , (2012).</li><li>Ogunro, O., Karunwi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chiral+Asymmetry+of+Helical+Polymer+Nanowire.">Chiral Asymmetry of Helical Polymer Nanowire.</a> The Journal of Physical Chemistry Letters. 1, 704-707 (2010).</li></ol>

No conflicts of interest declared.

In this report, we have presented a powerful approach for preparing biofunctional nanofibers. The nanofibers are decorated to a functional group which is specific to a model protein. The procedure and approach reported in this communication is general in nature and may be used to prepare nanofibers decorated with any functional group desired. The anionic living polymerization is powerful method to synthesize controlled polymer structures covalently connected to any number of interesting functional or functional groups wh...

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>Sodium Metal</td>
 <td>Sigma-Aldrich</td>
 <td>282065</td>
 </tr>
 <tr>
 <td>Benzophenone</td>
 <td>Sigma-Aldrich</td>
 <td>239852 </td>
 </tr>
 <tr>
 <td>2-methoxystyrene</td>
 <td>Sigma-Aldrich</td>
 <td>563064 </td>
 </tr>
 <tr>
 <td>Tetrahydrofuran</td>
 <td>Sigma-Aldrich</td>
 <td>178810 </td>
 </tr>
 <tr>
 <td>Chlorobenzene</td>
 <td>Sigma-Aldrich</td>
 <td>319996 </td>
 </tr>
 <tr>
 <td>Single walled CNTs</td>
 <td>Sigma-Aldrich</td>
 <td>704113 </td>
 </tr>
 <tr>
 <td>Polystyrene </td>
 <td>Sigma-Aldrich</td>
 <td>81416</td>
 </tr>
 <tr>
 <td>Silicon Wafers</td>
 <td>Silicon Quest Int'l</td>
 <td>720200</td>
 </tr>
 <tr>
 <td>Zeiss FESEM</td>
 <td>Carl Zeiss Inc.</td>
 <td>Ultra 60</td>
 </tr>
 <tr>
 <td>Probestation with Bausch &amp; Lomb MicroZoom II High Performance Microscope</td>
 <td>Bausch and Lomb</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Leica Scanning Confocal System</td>
 <td>Leica Microsystems</td>
 <td>TCS SP2</td>
 </tr>
 <tr>
 <td>Sub-femtoamp Remote Sourcemeter</td>
 <td>Keithley Instruments</td>
 <td>6430 </td>
 </tr>
 <tr>
 <td>Autoranging Digital Multimeter</td>
 <td>Keithley Instruments</td>
 <td>175A </td>
 </tr>
 <tr>
 <td>Syringe Pump </td>
 <td>Chemyx Inc. </td>
 <td>Fusion 200 </td>
 </tr>
 <tr>
 <td>Zeiss Optical Microscope</td>
 <td>Carl Zeiss Inc.</td>
 <td>Zeiss/Axiotech </td>
 </tr>
 </tbody>
</table>

procedure for fabricating biofunctional nanofibers

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups may be utilized to study material-biomarker interactions i.e. act as biosensors with potential as single molecule detectors. We have developed an effective approach for preparing functional polymers where the functionality has the capacity of specifically binding with a model protein. In our model system, the functional group is 2,4-dinitrophenyl (DNP) and the protein is anti-DNP IgE (Immunoglobulin E). The functional polymer, &alpha;,&omega;-bi[2,4-dinitrophenyl caproic][poly(ethylene oxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP), is prepared by anionic living polymerization. The difunctional initiator utilized in the polymerization was prepared by electron transfer reaction of &alpha;-methylstyrene and potassium (mirror) metal. The 2-methoxystyrene monomer was added first to the initiator, followed by the addition of the second monomer, ethylene oxide, and finally the living polymer was terminated by methanol. The &alpha;,&omega;-dihydroxyl polymer [HO-PEO-P2MS-PEO-OH] was reacted with N-2,4-DNP-&isin;-amino caproic acid, by DCC coupling, resulting in the formation of &alpha;,&omega;-bi[2,4-dinitrophenylcaproic][poly(ethyleneoxide)-b-poly(2-methoxystyrene)-b-poly(ethylene oxide)] (CDNP-PEO-P2MS-PEO-CDNP). The polymers were characterized by FT-IR, 1H NMR and Gel Permeation Chromatography (GPC). The molecular weight distributions of the polymers were narrow (1.1-1.2) and polymers with molecular weights greater than 50,000 was used in this study. The polymers were yellow powders and soluble in tetrahydrofuran. A water soluble CDNP-PEO-P2MS-PEO-CDNP/ DMEG (dimethoxyethylene glycol) complex binds and achieves steady state binding with solution IgE within a few seconds. Higher molecular weight (water insoluble i.e. around 50,000) CDNP-PEO-P2MS-PEO-CDNP polymers, containing 1% single wall carbon nanotubes (SWCNT) were processed into electroactive nanofibers (100 nm to 500 nm in diameter) on silicon substrate. Fluorescence spectroscopy shows that anti-DNP IgE interacts with the nanofibers by binding with the DNP functional groups decorating the fibers. These observations suggest that appropriately functionalized nanofibers hold promise for developing biomarker detection device.

Watch this Scientific Journal Video about Procedure for Fabricating Biofunctional Nanofibers at JoVE.com

Procedure for Fabricating Biofunctional Nanofibers

An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Electrospinning is an effective processing method for preparing nanofibers decorated with functional groups. Nanofibers decorated with functional groups ...

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12.5K Views.  Clark Atlanta University. An efficient approach for preparing nanofibers decorated with functional groups capable of specifically interacting with proteins is described. The approach first requires the preparation of a polymer functionalized with the appropriate functional group. The functional polymer is fabricated into nanofibers by electrospinning. The effectiveness of the binding of the nanofibers with a protein is studied by confocal microscopy.

Video: Procedure for Fabricating Biofunctional Nanofibers

A Craniotomy Surgery Procedure for Chronic Brain Imaging

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics),  to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an ...

Murine Renal Transplantation Procedure

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell et al over 30 years ago (1) and refined by Zhang et al years later (2), few people in the world have mastered this procedure. In our laboratory we have successfully performed 1200 orthotopic kidney transplantations with &gt; 90% survival rate. The key points for success include stringent control of reperfusion injury, bleeding and thrombosis, both during the procedure and post-transplantation, and use of 10-0 instead of 11-0 suture for anastomoses.
Post-operative care and treatment of the recipient is extremely important to transplant success and evaluation. All renal graft recipients receive antibiotics in the form of an injection of penicillin immediately post-transplant and sulfatrim in the drinking water continually. Overall animal health is evaluated daily and whole blood creatinine analyses are performed routinely with a portable I-STAT machine to assess graft function.

Renal transplantation in mice is a technically challenging procedure that requires careful post-operative care and treatment for success.

Renal orthotopic transplantation in mice is a technically challenging procedure. Although the first kidney transplants in mice were performed by Russell ...

Bioluminescence Imaging of Heme Oxygenase-1 Upregulation in the Gua Sha Procedure

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol for bioluminescent optical imaging of HO-1-luciferase transgenic mice reported in this manuscript provides a rapid in vivo assay of the upregulation of the heme oxygenase-1 (HO-1) gene expression in response to the Gua Sha procedure. HO-1 has long been known to provide cytoprotection against oxidative stress. The upregulation of HO-1, assessed by the bioluminescence output, is thought to represent an antioxidative response to circulating hemoglobin products released by Gua Sha. Gua Sha was administered by repeated strokes of a smooth spoon edge over lubricated skin on the back or other targeted body part of the transgenic mouse until petechiae (splinter hemorrhages) or ecchymosis (bruises) indicative of extravasation of blood from subcutaneous capillaries was observed. After Gua Sha, bioluminescence imaging sessions were carried out daily for several days to follow the dynamics of HO-1 expression in multiple internal organs.

Gua Sha, traditional Chinese therapeutic skin scraping, causes subcutaneous microvascular blood extravasation. We report a protocol of bioluminescence imaging of HO-1-luciferase transgenic mice to demonstrate that Gua Sha upregulates heme oxygenase-1 (HO-1) in multiple organs.

Gua Sha is a traditional Chinese folk therapy that employs skin scraping to cause subcutaneous microvascular blood extravasation and bruises. The protocol ...

Mammary Epithelial Transplant Procedure

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, followed by the mammary epithelial transplant procedure. The mammary transplant procedure is widely used by mammary biologists because it takes advantage of the fact that significant development of the mammary epithelium doesn't occur until after puberty. At 3 weeks of age, growth of the mammary epithelial tree is confined to the vicinity of the nipple and the fat pad is largely devoid of mammary epithelium, but by 7 weeks of age the epithelial ductal tree extends throughout the entire fat pad. Therefore, if this small portion of the fat pad containing epithelium, the region between the nipple and the lymph node, is removed at 3 weeks of age, the endogenous epithelium will never populate the mammary fat pad and the fat pad is described as "cleared". At this time, mammary epithelium from another source can be transplanted in the cleared fat pad where it has the potential to extend mammary ductal trees through out the fat pad. This procedure has been utilized in many experimental models including the examination of tumor phenotype in transgenic mammary epithelial tissue without the confounding effects of genotype on the entire animal3, in the identification of mammary stem cells by transplanting cells in limited dilution4,5, determining if hyperplastic nodules proceed to mammary tumors6, and to assess the effect of prior hormone exposure on the behavior of the mammary epithelium7,8.
Three week old host mice are anesthetized, cleaned and restrained on a surgical stage. A mid-sagittal incision is made through the skin, but not the peritoneum, extending from the pubis to the sternum. Oblique cuts are made through the skin from the mid-sagittal incision across the pelvis toward each leg. The skin is pulled away from the peritoneum to expose the 4th inguinal mammary gland. The fat pad is cleared by removing the fat pad tissue anterior to the lymph node. Epithelium fragments or epithelial cells are transplanted into the remaining cleared fat pad and the mouse is closed.

This article demonstrates the procedure developed by DeOme KB et al. (1959) and the sparing procedure developed by Brill B et al. (2008) for clearing the 4th inguinal mammary fat pad of a pubescent mouse in preparation for transplantation of mammary fragments, mammary epithelial cells, or mammary tumor cells.

This article describes and compares the fat pad clearance procedure developed by DeOme KB et al.1 and the sparing procedure developed by Brill B et al.2, ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Isolation of Human Hepatocytes by a Two-step Collagenase Perfusion Procedure

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. As such, hepatocytes are an important model for a large variety of research questions. In particular, the use of human hepatocytes is especially important in the fields of pharmacokinetics, toxicology, liver regeneration and translational research. Thus, this method presents a modified version of a two-step collagenase perfusion procedure to isolate hepatocytes as described by Seglen 1.
Previously, hepatocytes have been isolated by mechanical methods. However, enzymatic methods have been shown to be superior as hepatocytes retain their structural integrity and function after isolation. This method presented here adapts the method designed previously for rat livers to human liver pieces and results in a large yield of hepatocytes with a viability of 77&plusmn;10%. The main difference in this procedure is the process of cannulization of the blood vessels. Further, the method described here can also be applied to livers from other species with comparable liver or blood vessel sizes.

A modified two-step collagenase perfusion procedure for isolation of human hepatocytes is described. This method can also be applied to other mammalian livers. The isolated hepatocytes are available in high yield and viability, making them a suitable model for scientific research in areas such as liver regeneration, pharmacokinetics and toxicology.

The liver, an organ with an exceptional regeneration capacity, carries out a wide range of functions, such as detoxification, metabolism and homeostasis. ...

Procedure to Evaluate the Efficiency of Flocculants for the Removal of Dispersed Particles from Plant Extracts

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used as manufacturing platforms for added-value products such as biopharmaceuticals. In both cases, liquid plant extracts may need to be clarified to remove particulates. Optimal clarification reduces the costs of filtration and centrifugation by increasing capacity and longevity. This can be achieved by introducing charged polymers known as flocculants, which cross-link dispersed particles to facilitate solid-liquid separation. There are no mechanistic flocculation models for complex mixtures such as plant extracts so empirical models are used instead. Here a design-of-experiments procedure is described that allows the rapid screening of different flocculants, optimizing the clarification of plant extracts and significantly reducing turbidity. The resulting predictive models allow the identification of robust process conditions and sets of polymers with complementary properties, e.g. effective flocculation in extracts with specific conductivities. The results presented for tobacco leaf extracts can easily be adapted to other plant species or tissues and will thus facilitate the development of more cost-effective downstream processes for commodities and plant-derived pharmaceuticals.

The design-of-experiments procedure presented here allows the evaluation of different flocculants in terms of their ability to aggregate dispersed particles in plant extracts, thus reducing turbidity and the costs of downstream processing.

Plants are important to humans not only because they provide commodities such as food, feed and raw materials, but increasingly because they can be used ...

Procedure and Key Optimization Strategies for an Automated Capillary Electrophoretic-based Immunoassay Method

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. However, similar to other antibody-based protein assays, optimization of capillary-based immunoassay parameters, such as protein concentration, antibody dilution, and exposure time is an important prerequisite to the generation of meaningful and reliable data. Measurements must fall within the linear range of the assay where changes in signal are directly proportional to changes in lysate concentration. The process of choosing appropriate lysate concentrations, antibody dilutions, and exposure times in the human bronchial epithelial cell line, BEAS-2B, is demonstrated here. Assay linearity is shown over a range of whole cell extract protein concentrations with p53 and &#945;-tubulin antibodies. An example of signal burnout is seen at the highest concentrations with long exposure times, and an &#945;-tubulin antibody dilution curve is shown demonstrating saturation. In addition, example experimental results are reported for doxorubicin-treated cells using optimized parameters.

A capillary-based immunoassay using a commercial platform to measure target proteins from total protein preparations is demonstrated. In addition, assay parameters of exposure time, protein concentration, and antibody dilution are optimized for a cell culture model system.

New technologies that utilize capillary-based immunoassays promise faster and more quantitative protein assessment compared to traditional immunoassays. ...

A Semiautomated ChIP-Seq Procedure for Large-scale Epigenetic Studies

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific histone modifications, such as H3K27ac, to help identify cis-regulatory DNA elements. The manual process to complete a ChIP-Seq is labor intensive, technically challenging, and often requires large-cell numbers (&#62;100,000 cells). The method described here helps to overcome those challenges. A complete semiautomated, microscaled H3K27ac ChIP-Seq procedure including cell fixation, chromatin shearing, immunoprecipitation, and sequencing library preparation, for batch of 48 samples for cell number inputs less than 100,000 cells is described in detail. The semiautonomous platform reduces technical variability, improves signal-to-noise ratios, and drastically reduces labor. The system can thereby reduce costs by allowing for reduced reaction volumes, limiting the number of expensive reagents such as enzymes, magnetic beads, antibodies, and hands-on time required. These improvements to the ChIP-Seq method suit perfectly for large-scale epigenetic studies of clinical samples with limited cell numbers in a highly reproducible manner.

This paper describes a semiautomated, microscaled ChIP-Seq protocol of DNA regions associated with a specific histone modification (H3K27ac) using a ChIP liquid-handler platform, followed by library preparation using tagmentation. The procedure includes control assessment for quality and quantity purposes and can be adopted to other histone modifications or transcription factors.

Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) is a powerful and widely used approach to profile chromatin DNA associated with specific ...

Enhancing Survival and Development of Marmosets Using a Hand-Rearing Approach

Hand-Rearing Method for Infant Marmosets

The common marmoset (Callithrix jacchus) is a small and highly social New World monkey with high reproduction rates, which has been proven to be a compelling non-human primate model for biomedical and neuroscience research. Some females give birth to triplets; however, the parents cannot raise all of them. To save these infants, we have developed a hand-rearing method for raising newborn marmosets. In this protocol, we describe the formula of the food, the time for feeding, the configuration of the temperature and humidity, as well as the adaptation of the hand-reared infants to the colony environment. This hand-rearing method significantly increases the survival rate of marmoset infants (without hand-rearing: 45%; with hand-rearing: 86%) and provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments. As the method is practical and easy to use, we anticipate that it could also be applied to other labs working with common marmosets.

Author Spotlight: Marmoset Research - Scope and Challenges 

Here, we describe a hand-rearing method for raising infant marmosets in an animal incubator. This method greatly increases the survival rate of marmoset infants, which provides the opportunity to study the development of marmoset infants with similar genetic backgrounds raised in different postnatal environments.