M.Z. and K.S.F. are supported by American Heart Association Scientist Development Award 17SDG33411180, and by a grant awarded under the Pitt Innovation Challenge (PInCh&#174;), through the Clinical and Translational Science Institute at the University of Pittsburgh.

The University of Pittsburgh&#39;s Institutional Animal Care and Use Committee approved all animal protocols specified in this publication prior to undertaking any of these animal experiments.
NOTE: This protocol details how to perform ex vivo biodistribution studies utilizing ex vivo optical fluorescent imaging followed by in vivo biodistribution studies by embedding the organs in paraffin, sectioning, and performing fluorescent microscopy. Although CTP is used as an example, this method can be applied to any fluorescently labeled CPP.
1. In vivo imaging
<ol>
	<li>Synthesize CTP using solid phase synthesis techniques9,10 from a peptide synthesis facility using L-amino acids with an N-terminus labeled with Cy5.5 and C-terminus amide-capped for increased stability. 
	NOTE: All synthesized CPPs should be characterized using high-performance liquid chromatography (HPLC) and/or matrix assisted laser desorption/ionization (MALDI) prior to use11.</li>
	<li>Make a 1 mM or 10 mM stock solution of CTP in dimethyl sulfoxide (DMSO), aliquot, and store at -80 &#176;C, light-protected. 
	NOTE: Peptides are delivered as lyophilized powder. Lyophilized powder can be stored long-term (6 months-2 years) in -20 &#176;C, light-protected, and under hygroscopic conditions. When aliquoted in DMSO and stored, freeze-thaw cycles should be avoided.</li>
	<li>Weigh and anesthetize 6-week-old CD1 female mice using 2 &#181;L/g of tissue weight of a ketamine/xylazine solution (100 mg/kg ketamine and 20 mg/kg xylazine) administered intramuscularly (hind leg) or intraperitoneally (lower left quadrant). 
	NOTE: Ketamine/xylazine solutions should be made fresh on the day of use. Unused ketamine/xylazine should be disposed of appropriately and volumes used versus volumes discarded recorded in a log, as ketamine is a controlled substance. Adequate level of anesthesia will be achieved in 5&#8722;7 min, as assessed by lack of response to a toe pinch.</li>
	<li>Calculate a 10 mg/kg dose of Cy5.5-CTP, dilute with phosphate buffered saline (PBS) to no more than 200 &#181;L, and inject either retro-orbitally or through tail vein injection using an insulin syringe (Figure 1A).</li>
	<li>Allow peptide to circulate for the prespecified time, ranging from 15 min-6 h.</li>
	<li>Euthanize the mouse using the high CO2 inhalational method as specified by the Institutional Animal Care and Use Committee and open the chest cavity using scissors (Figure 1B).</li>
	<li>Use scissors to place a small nick in the lateral, free wall of the right atrium and inject 3 mL of 10% buffered formalin phosphate using a 26 G needle for the dual purpose of perfusion fixing the organs of the mouse and flushing out red blood cells (Figure 1C).</li>
	<li>Dissect out heart, lung, liver, kidneys, spleen, large intestines, small intestines, bladder, ovaries/testes, and brain and place into individual wells of a 12 well plate for ex vivo optical imaging (Figure 1D).</li>
	<li>Start the image acquisition software and click the Initialize button to prepare the system for imaging samples. Open the imaging wizard, select fluorescence, then filter pair, then select the Cy5.5 dye from the list of dyes to apply 580 nm excitation and 620 nm emission filters. Select manual settings for exposure parameters then set the stage position to B, set exposure time to 1 second, set the F/stop to 8, and set the binning to small. 
	NOTE: The excitation emission filters will vary depending on the label used. The excitation and emission of the label used can be manually entered for the software to assign excitation and emission filters. Ensure that the counts are between 600-60,000 in order to avoid saturation. Depending on the system being used, automatic acquisition optimization might be available. If the system has compensation to compare images acquired with different settings, the auto settings can be used. If no compensation is available, settings will need to be determined, saved, and used consistently across samples.</li>
	<li>When the system is completely initialized, arrange the organs of interest in a 12 well plate and place it inside of the optical imaging chamber, ensuring that the samples are arranged as desired for the images. Select acquire, then select a save location for the images. Information about the mouse can be written into the edit image labels pop-up window.</li>
	<li>After imaging, remove the samples from the chamber and store in 10% buffered formalin phosphate in a volume at least 20 times the volume of the tissue in scintillation vials with light protection at room temperature (RT).</li>
	<li>Quantify the images by setting units to Radiant Efficiency then selecting the 4x3 ROI and adjusting to fit each well into one square of the ROI evenly, followed by clicking measure. Open the grid ROI measurements tab, select export, and save the measurements as a .cvs file.</li>
</ol>
2. Histology
NOTE: Follow steps 1.1&#8722;1.8 to obtain different organs. Alternatively, the same organs that were imaged can be placed in formalin immediately and used for sectioning as detailed below.
<ol>
	<li>Store each organ individually in 10% buffered formalin phosphate for a minimum of 48 h before processing for histology.</li>
	<li>When the organs are sufficiently fixed after 48 h, transfer the organs into tissue processing and embedding cassettes and place into a tissue processing machine (Figure 1E).</li>
	<li>Set the processing machine to dehydrate the tissue in 70% ethanol for 30 min, followed by 80% ethanol for 30 min, 95% ethanol for 30 min, 95% ethanol for 30 min, 100% ethanol for 15 min, 100% ethanol for 20 min, and finally 100% ethanol for 20 min.</li>
	<li>Clear the tissue in xylene 2x, with 30 min for each xylene treatment.</li>
	<li>Infiltrate the cleared tissue with paraffin wax 4x at 60 &#176;C for 30 min with each treatment.</li>
	<li>Embed in paraffin using metal molds. Fill the mold with molten paraffin (kept at 65 &#176;C), and transfer to a cold plate. As the paraffin at the bottom of the mold begins to solidify, place the organ into the paraffin (Figure 1F).</li>
	<li>Place a labeled cassette on top of the mold as a backing and overfill with molten paraffin. Allow to cool until solid. Then store the block in a -20 &#176;C freezer overnight, allowing the wax to shrink further for easy removal from the molds. 
	NOTE: The blocks can now be stored at RT with light protection.</li>
	<li>Prepare a 38 &#176;C water bath with distilled water. Set up the microtome with a blade angle of 6&#176; and a section thickness of 10 &#181;m.</li>
	<li>Mount the tissue blocks in the microtome and begin cutting until sections containing tissue are obtained. Then place the blocks face down in the water for 5 min or until the tissue has absorbed some moisture (e.g., when a thin white outline of the tissue appears in the block [Figure 1G]). 
	NOTE: The blade should be replaced frequently to ensure the quality of the sections.</li>
	<li>Place the tissue onto a flat ice block for 10 min, then return it to the microtome in the same orientation as in step 2.9. Begin taking sections and allow truncated sections to form long ribbons of 6&#8722;10 sections each. Discard suboptimal paraffin ribbons until a high-quality ribbon of sufficient length to cover a slide is produced. Optimize sectioning by adjusting cutting speeds, tissue moisturization, and water bath temperature in order to avoid shearing or wrinkling of the tissue sections. 
	NOTE: Sections that have holes where tissue fell out, rips in the tissue, or tight wrinkles and folds in the tissue should be discarded.</li>
	<li>Carefully pick the quality ribbon of choice with blunt edge forceps and float on the surface of the 38 &#176;C water bath. Let the sections sit on the surface until they smooth out, taking care to not leave them too long to prevent the paraffin from disintegrating and tearing the section apart.</li>
	<li>Float the flattened sections onto the surface of clean glass slides. Place the slides into a 65 &#176;C oven for 30 min to melt the wax. 
	NOTE: These slides can be stored at RT with light protection.</li>
	<li>Deparaffinize the slides in xylene 3x, 10 min for each treatment.</li>
	<li>Rehydrate the tissue in 100% ethanol for 5 min, followed by 95% ethanol for 5 min, 70% ethanol for 5 min, 50% ethanol for 5 min, and finally 1x tris-buffered saline (TBS) for 5 min (Figure 1H). Allow the slides to dry overnight. 
	NOTE: These slides can be stored long-term at RT as long as they are light-protected.</li>
	<li>Mount the slides with coverslips using 125 &#181;L of mounting media containing 4&#8242;,6-diamidino-2-phenylindole (DAPI), a nuclear fluorescent probe (Figure 1I). Dry slides overnight at RT, light-protected. 
	NOTE: Dried slides can be stored long-term at 4 &#176;C, light-protected.</li>
	<li>Image slides using fluorescent microscopy. 
	NOTE: Saturated images should be avoided, as they are not quantitatively useful. Adjusting the exposure and gain settings can be lowered to avoid saturation. Take care to use the same settings across the samples being compared. Avoid bleaching of the tissue by limiting exposure times.</li>
</ol>

<ol>
	<li>Green, M., Loewenstein, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autonomous+functional+domains+of+chemically+synthesized+human+immunodeficiency+virus+tat+trans-activator+protein.">Autonomous functional domains of chemically synthesized human immunodeficiency virus tat trans-activator protein.</a> Cell. 55 (6), 1179-1188 (1988).</li><li>Frankel, A. D., Pabo, C. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+uptake+of+the+tat+protein+from+human+immunodeficiency+virus.">Cellular uptake of the tat protein from human immunodeficiency virus.</a> Cell. 55 (6), 1189-1193 (1988).</li><li>Derossi, D., Joliot, A. H., Chassaing, G., Prochiantz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+third+helix+of+the+Antennapedia+homeodomain+translocates+through+biological+membranes.">The third helix of the Antennapedia homeodomain translocates through biological membranes.</a> Journal of Biological Chemistry. 269 (14), 10444-10450 (1994).</li><li>Schwarze, S. R., Ho, A., Vocero-Akbani, A., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+protein+transduction:+delivery+of+a+biologically+active+protein+into+the+mouse.">In vivo protein transduction: delivery of a biologically active protein into the mouse.</a> Science. 285 (5433), 1569-1572 (1999).</li><li>Zahid, M., Robbins, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+penetrating+peptides:+therapeutic+promises+and+challenges.">Cell-type specific penetrating peptides: therapeutic promises and challenges.</a> Molecules. 20 (7), 13055-13070 (2015).</li><li>Zahid, M., Robbins, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+tissue-specific+protein+transduction+domains+using+peptide+phage+display.">Identification and characterization of tissue-specific protein transduction domains using peptide phage display.</a> Methods in Molecular Biology. 683, 277-289 (2011).</li><li>Zahid, M., 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=Identification+of+a+cardiac+specific+protein+transduction+domain+by+in+vivo+biopanning+using+a+M13+phage+peptide+display+library+in+mice.">Identification of a cardiac specific protein transduction domain by in vivo biopanning using a M13 phage peptide display library in mice.</a> PLoS One. 5 (8), e12252 (2010).</li><li>Zahid, M., 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=Cardiac+Targeting+Peptide,+a+Novel+Cardiac+Vector:+Studies+in+Bio-Distribution,+Imaging+Application,+and+Mechanism+of+Transduction.">Cardiac Targeting Peptide, a Novel Cardiac Vector: Studies in Bio-Distribution, Imaging Application, and Mechanism of Transduction.</a> Biomolecules. 8 (4), E147 (2018).</li><li>Amblard, M., Fehrentz, J. A., Martinez, J., Subra, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+and+protocols+of+modern+solid+phase+Peptide+synthesis.">Methods and protocols of modern solid phase Peptide synthesis.</a> Molecular Biotechnology. 33 (3), 239-254 (2006).</li><li>Katritzky, A. R., Yoshioka, M., Narindoshvili, T., Chung, A., Johnson, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+labeling+of+peptides+on+solid+phase.">Fluorescent labeling of peptides on solid phase.</a> Organic and Biomolecular Chemistry. 6 (24), 4582-4586 (2008).</li><li>Prabhala, B. K., Mirza, O., Hojrup, P., Hansen, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Synthetic+Peptides+by+Mass+Spectrometry.">Characterization of Synthetic Peptides by Mass Spectrometry.</a> Methods in Molecular Biology. 1348, 77-82 (2015).</li><li>Arms, L., 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=Advantages+and+Limitations+of+Current+Techniques+for+Analyzing+the+Biodistribution+of+Nanoparticles.">Advantages and Limitations of Current Techniques for Analyzing the Biodistribution of Nanoparticles.</a> Frontiers in Pharmacology. 9, 802 (2018).</li><li>Leblond, F., Davis, S. C., Valdes, P. A., Pogue, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-clinical+whole-body+fluorescence+imaging:+Review+of+instruments,+methods+and+applications.">Pre-clinical whole-body fluorescence imaging: Review of instruments, methods and applications.</a> Journal of Photochemistry and Photobiology. 98 (1), 77-94 (2010).</li><li>Hughes, L. D., Rawle, R. J., Boxer, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Choose+your+label+wisely:+water-soluble+fluorophores+often+interact+with+lipid+bilayers.">Choose your label wisely: water-soluble fluorophores often interact with lipid bilayers.</a> PLoS One. 9 (2), e87649 (2014).</li><li>McGowan, J. W., Bidwell, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Use+of+Ex+Vivo+Whole-organ+Imaging+and+Quantitative+Tissue+Histology+to+Determine+the+Bio-distribution+of+Fluorescently+Labeled+Molecules.">The Use of Ex Vivo Whole-organ Imaging and Quantitative Tissue Histology to Determine the Bio-distribution of Fluorescently Labeled Molecules.</a> Journal of Visualized Experiments. (118), e54987 (2016).</li><li>Waters, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+and+precision+in+quantitative+fluorescence+microscopy.">Accuracy and precision in quantitative fluorescence microscopy.</a> Journal of Cell Biology. 185 (7), 1135-1148 (2009).</li></ol>

M.Z. and Paul D. Robbins (University of Minnesota, Minnesota, MN, USA) hold a patent on the use of cardiac targeting peptide as a cardiac vector (Cardiac-specific protein targeting domain, U.S. Patent Serial No. 9,249,184). M.Z. also serves as Chief Scientific Officer and is on the Board of Directors of the startup Vivasc Therapeutics Inc. and holds substantial future equity in it.

Animal models are essential for preclinical drug development at every stage of the process from identification of novel CPPs, biodistribution studies, mechanism of transduction, to ultimately testing for efficacy of the delivered cargo using these novel CPPs as vectors. There are many commonly used methods available to assess biodistribution such as histology, nuclear medicine imaging (SPECT and PET), and in vivo optical imaging12. Nuclear imaging methods can be cumbersome due to the limited availability of small animal SPECT and PET systems, as well as the ability to produce radiolabel-drug conjugates, which require the expertise of a radiochemist. In contrast, fluorescent labels are much simpler and can be cost effective to work with. The protocol described in this paper allows for rapid analysis of biodistribution using multiple methods. Ex vivo whole organ fluorescent optical imaging allows for the immediate comparison of fluorescence across different tissues and treatment groups and can identify organ uptake and time to peak uptake in organ of interest in a semiquantitative manner. Quantitative tissue histology requires more extensive processing to treat, section, image, and analyze tissues, but it provides more data on the microscopic level and is a current standard technique. It is worthwhile to note that direct, quantitative comparison across the two techniques is not possible, because the autofluorescence seen with each technique for different organs and excitation wavelengths varies significantly.
An important part of this protocol is selecting the right fluorophore to label candidate CPPs for experiments. One potential issue is spectra overlap, which can be problematic when multiple fluorophores are needed. The DAPI fluorescent mounting media and Cy5.5 do not have overlapping spectra. However, for certain applications where multiple fluorophores are needed, the risk of spectral overlap needs to be carefully considered. Depending on the system being used, fluorophore selection may be limited. Therefore, knowledge of the system&#39;s capabilities is key. Fluorescent optical systems are best utilized with far-red or near-infrared fluorophores due to the high tissue absorption of shorter wavelengths13. Fluorophores in the range of enhanced green fluorescent protein have a major limitation, because there is significant organ autofluorescence seen at its excitation wavelength, specifically in brain and liver tissue. Depending on the conditions of an experiment, some fluorophores are best avoided. Some water-soluble organic fluorophores have a strong interaction with lipid bilayers, which can cause false positives. Hence, taking steps to determine if a fluorophore has strong affinity to the tissue of interest is advisable14. Another factor to consider is selecting the appropriate method of fluorophore conjugation, which can be an important parameter affecting the results. CPPs can be labeled fluorescently at the N- or C-terminus through a covalent bond between the N-terminus of the peptide and the carboxyl group of the dye such as Cy5.5-NHS. Care should be taken, because the mechanism of transduction of most CPPs is not understood in detail and conjugation at one end may affect the uptake mechanism more so than at the other end. Another possibility for labeling CPPs is through biotinylating the N-terminus for conjugation to fluorescently labeled streptavidin. Using this strategy has the convenience of allowing different fluorescent streptavidin conjugates to be utilized. However, a possible limitation of this strategy is that a biotin-streptavidin complex is a large construct, which could potentially interfere with transduction.
Fluorescent optical imaging systems are an effective strategy for generating comparison of fluorescence across different organs and treatments efficiently but are incapable of producing a quantitative measure of absolute concentrations in tissue. This is due to light scattering effects within the tissue, which is further compounded by the naturally occurring variety in tissue sizes and densities, and differences in vascularity, with variable fluorescence scattering. Tissue autofluorescence can be a factor as well, due to naturally occurring biochemical sources such as collagen, or dietary sources like chlorophyll in food13.
Histology is the most commonly used method of measuring biodistribution and can potentially be used to accurately measure and compare uptake across different tissues over time. Light scattering issues are avoided using this method because all tissues are sectioned to the same thickness15. A major advantage of this method is the ability to include additional fluorescent labels postsectioning for immunohistochemistry. Although the addition of another fluorophore could make imaging more challenging, the use of fluorescent labels can be useful for localization of a CPP to particular intracellular compartments, like lysosomes or mitochondria. An antibody could be used in a confocal microscopy experiment to determine if a transduced candidate CPP colocalizes with a structure of interest, which can show the potential of a CPP as a delivery agent. One limitation of this method is that preparation of slides from organ samples can be time consuming, labor intensive, and prone to human error12,15. When imaging slides, care should be taken to not image the same location for too long to avoid photobleaching. Some photobleaching will be inevitable, depending on the sensitivity of the fluorophore. Care should be taken at every step of this protocol to protect the samples from ambient light and store them properly16. We recommend that slides be stored at 4 &#176;C, light-protected, for future imaging.
There are a variety of methods available for measuring the biodistribution of a candidate CPP that require specialized equipment and can produce comparable results, though they may require more complex CPP labeling. The protocol described in this paper uses two compatible methods to efficiently produce biodistribution data in the context of a living system while allowing for the acquisition of greater in-depth information about peptide internalization within cells from the same sample, thus cutting the number of animals needed for a study by half. These methods were used to generate the above data, which demonstrate that both methods can be utilized sequentially in the same animal, and the quality of the data generated by each. Our results also highlight the inability to directly correlate results between the two techniques in a quantitative manner.

The cell plasma membrane is a semipermeable barrier that is essential for cell integrity and survival and serves to regulate the interior of the cell by controlling movement of substances into the cell. Although vital for survival, it also presents a barrier to delivery of cargo to the cell. In 1988, the trans-activator of transcription (Tat) protein of the human immunodeficiency virus was shown to enter cultured cells and promote viral gene expression1,2, with the domains responsible for this transduction limited to an arginine and lysine rich 13-amino acid region of the third helix3 These were thus named cell penetrating peptides (CPPs). This was followed by research showing the ability of the Tat peptide to carry functional &#946;-galactosidase into multiple cell types4. Since the initial description, the number of cell-penetrating peptides has increased dramatically. These transduction domains are naturally occurring or synthetic short peptides, typically 6&#8722;20 amino acids long, that are able to carry functional cargoes across cell membranes. These cargoes can range from other small peptides, full-length proteins, nucleic acids, nanoparticles, viral particles, fluorescent molecules, and radioisotopes5. The initial CPPs described were not cell specific, with Tat being taken up by multiple cell types and even crossing the blood-brain barrier4, hence limiting its therapeutic potential. In order to identify cell-specific CPPs, investigators have undertaken phage display utilizing large, commercially available phage libraries6. Our own work using a combinatorial in vitro and in vivo phage display methodology led to the identification of a CPP named cardiac targeting peptide (CTP)7, a 12-amino acid, nonnaturally occurring peptide (NH2-APWHLSSQYSRT-COOH) that targets the heart with peak uptake at 15 min after a peripheral intravenous injection8. Using immunofluorescent colocalization with actin, an intracellular marker, and exclusion of laminin, a cell membrane marker, we showed that CTP is internalized into mouse cardiomyocytes after an intravenous injection7. Additionally, we incubated human induced pluripotent stem cell-derived beating cardiomyocytes with dual-labeled CTP, labeled with 6-carboxyfluorescein at its C-terminus and rhodamine at its N-terminus through an ester linkage that could only be cleaved off by intracellular esterases. Rapid accumulation of rhodamine into beating cardiomyocytes was observed within 15 min on confocal microscopy8.
In this article, we present two complimentary methodologies that can be used to track biodistribution and confirm tissue-specific internalization of CPPs using CTP as an example. For these methodologies, CTP was synthesized, fluorescently labeled at the N-terminus with cyanine5.5 (CY5.5), and amide-capped at the C-terminus for greater peptide stability. The two methodologies used are in vivo fluorescent optical imaging systems and fluorescent microscopy of tissue sections. Both methods are very useful in studying biodistribution, uptake, and elimination of fluorescently labeled CPPs. The advantage of assessing biodistribution using these methods over others, such as single-photon emission tomography (SPECT) or positron emission tomography (PET), is that there is no need for the time-intensive radiolabeling of CPPs compared with fluorescent labeling, which is relatively easy and in routine use at all peptide synthesis facilities. The use of in vivo imaging quickly produces biodistribution data in the context of a living system, while sectioning provides greater in-depth information about peptide uptake and localization within cells via the preservation of morphological detail. This protocol can be applied to a wide variety of organs and tissues such as the heart, lung, liver, kidney, brain, spleen, stomach, large intestine, small intestine, skeletal muscle, bone, testes/ovaries, and eyes.

&#160; &#160;

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10% Buffered Formalin Phosphate</td>
			<td>ThermoFisher</td>
			<td>SF100-4</td>
			<td></td>
		</tr>
		<tr>
			<td>10x Tris Buffered Saline (TBS)</td>
			<td>ThermoFisher</td>
			<td>BP2471-1</td>
			<td></td>
		</tr>
		<tr>
			<td>12-well Cell Culture Plate</td>
			<td>ThermoFisher</td>
			<td>353043</td>
			<td></td>
		</tr>
		<tr>
			<td>1x TBS Solution</td>
			<td></td>
			<td></td>
			<td>10x TBS is diluted 1:10 with deionized water. This solution can be stored at room temperature.</td>
		</tr>
		<tr>
			<td>20 mL Scintillation Vials</td>
			<td>Wheaton</td>
			<td>334173</td>
			<td></td>
		</tr>
		<tr>
			<td>26G Needles</td>
			<td>Becton Dickinson</td>
			<td>305110</td>
			<td></td>
		</tr>
		<tr>
			<td>28G 0.5ml Insulin syringes</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
			<td></td>
		</tr>
		<tr>
			<td>3mL Syringe</td>
			<td>Becton Dickinson</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>CD1 Mice</td>
			<td>Charles River</td>
			<td>022</td>
			<td>6 to 12 week old albino, female mice</td>
		</tr>
		<tr>
			<td>Cover Glass</td>
			<td>ThermoFisher</td>
			<td>12-544-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy5.5-CTP-amide</td>
			<td></td>
			<td></td>
			<td>Prepared by peptide synthesis, conjugated with Cy5.5 fluorophore, and purified using HPLC. Lyophilized powder is reconstituted in DMSO at 10mM concentration. After reconstituting, store at -80 &#176;C.</td>
		</tr>
		<tr>
			<td>Dapi Fluoromount G</td>
			<td>SouthernBiotech</td>
			<td>0100-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps</td>
			<td>Fine Science Tools</td>
			<td>99150-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Extra Fine Bonn Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine HCl 100mg/mL</td>
			<td>KetaVed</td>
			<td>NDC 50989-161-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine/Xylazine Solution</td>
			<td></td>
			<td></td>
			<td>Ketamine HCl is mixed with Xylazine 1:1 to produce a stock solution containing 50mg/mL ketamine and 10mg/mL Xylazine. This solution is made fresh before each use.</td>
		</tr>
		<tr>
			<td>Leica RM2235 Rotary Microtome</td>
			<td>Leica</td>
			<td>RM2235</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Slides</td>
			<td>ThermoFisher</td>
			<td>B9992000TN</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraplast X-TRA</td>
			<td>Sigma</td>
			<td>P3808-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Perkin Elmer Lumina S5 IVIS</td>
			<td>Perkin Elmer</td>
			<td>CLS148588</td>
			<td></td>
		</tr>
		<tr>
			<td>Sparkle Optical Lens Cleaner</td>
			<td>ThermoFisher</td>
			<td>NC0090079</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Processing/Embedding Cassettes</td>
			<td>ThermoFisher</td>
			<td>NC9499605</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek VIP Processing machine</td>
			<td>Tissue-Tek</td>
			<td>VIP 5A-F1</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>AnaSed</td>
			<td>NDC 59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylenes</td>
			<td>ThermoFisher</td>
			<td>X5-4</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo imaging of transduction efficiencies of cardiac targeting peptide

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest in developing these peptides, especially cell-specific ones, as novel vectors for delivering diagnostic and therapeutic materials. Our past work involving phage display identified a novel, nonnaturally occurring, 12 amino acid-long peptide that we named cardiac targeting peptide (CTP) due to its ability to transduce normal heart tissue in vivo with peak uptake seen in as little as 15 min after an intravenous injection. We have undertaken detailed biodistribution studies by injecting CTP labeled with fluorophore cyanine5.5, allowing it to circulate for various periods of time, and euthanizing, fixing, and sectioning multiple organs followed by fluorescent microscopy imaging. In this publication, we describe these processes as well as ex vivo imaging of harvested organs using an in vivo imaging system in detail. We provide detailed methodologies and practices for undertaking transduction as well as biodistribution studies using CTP as an example.

Using this protocol, we treated three mice with a 10 mg/kg dose of Cy5.5-CTP through a retro-orbital injection (Figure 1A). After allowing the peptide to circulate for 15 min, the mice were euthanized using a CO2 chamber, the chest opened through a median sternotomy incision, the right atrium was nicked, and the mice perfusion fixed using 3 mL of 10% phosphate-buffered formalin (Figure 1B,C). After fixation, the heart, lungs, liver, kidney, spleen, and brain were dissected out and arranged in a 12 well plate for ex vivo optical fluorescent imaging (Figure 1D and Figure 2A). Three control mice with no injections were also perfused, dissected, and imaged (Figure 1B&#8722;D and Figure 2B). The fluorescence data acquired for each set of organs can be compared due to the counts being converted to radiant efficiency, the relative fluorescence unit of the imaging software. This data can be quantified to yield both qualitative images and quantitative data for each organ in question (Figure 2C) across different time points for biodistribution studies. The autofluorescence of different organs in response to different excitation wavelengths is demonstrated in Figure 3A&#8722;E. Shorter excitation wavelengths, such as enhanced green fluorescent protein (EGFP), are associated with higher autofluorescence, especially in liver and brain, than far-red (Cy5.5) or near infrared (Cy7) excitation wavelengths (Figure 3). The imaged organs were fixed in 10% phosphate buffered formalin at RT for a minimum of 48 h, followed by a transfer to tissue processing and embedding cassettes (Figure 1E). The organs were processed and paraffinized in a tissue processing machine, positioned in molds filled with paraffin, frozen on a -20 &#176;C stage, and stored overnight at -20 &#176;C (Figure 1F). The samples were sectioned (Figure 1G) and treated with solution exchanges to rehydrate the tissue (Figure 1H). The prepared slides were then mounted with DAPI fluorescent mounting medium (Figure 1I). Once dry, usually overnight, the slides were imaged using fluorescent microscopy. Representative images of each organ from a mouse are shown in Figure 4A. The images from different organs were acquired using identical settings to allow for comparison across mice, and to allow for quantification (Figure 4B).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60895/60895fig1.jpg" /> 
Figure 1: Harvesting mouse organs for ex vivo optical fluorescent imaging and sectioning. (A) Wild type mouse injected retro-orbitally with CTP-Cy5.5. (B) Mouse dissected with chest opened, right atrium snipped, for perfusion fixation. (C) Heart injected with 3 mL of 10% buffered formalin phosphate for perfusion fixation of the animal. (D) Organs of interest harvested and arranged in a 12 well plate for fluorescent optical imaging. (E) Heart loaded into a cassette and processed using a tissue processor. (F) Heart embedded in paraffin. (G) Heart sectioned on a microtome. (H) Slides deparaffinized through a series of solution exchanges. (I) Sections mounted with coverslips using DAPI. <a href="https://www.jove.com/files/ftp_upload/60895/60895fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60895/60895fig2v2.jpg" /> 
Figure 2: Representative images from treated and control organs. (A) Organs from a mouse treated with 10 mg/kg CTP-Cy5.5. (B) Organs from an untreated control mouse. (C) Quantification of fluorescence for each set of organs. <a href="https://www.jove.com/files/ftp_upload/60895/60895fig2v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60895/60895fig3v2.jpg" /> 
Figure 3: Untreated mouse organs imaged at different excitation emission wavelengths to demonstrate how longer wavelengths produce less autofluorescence. (A) Cy7: 740&#8722;790. (B) Cy5.5: 660&#8722;710. (C) Cy5: 620&#8722;670. (D) Cy3: 520&#8722;570. (E) EGFP: 480&#8722;520. <a href="https://www.jove.com/files/ftp_upload/60895/60895fig3v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60895/60895fig4.jpg" /> 
Figure 4: Transduction of heart, lung, liver, and kidney after intravenous injection in mice. Wild type mice were injected with 10 mg/kg of Cy5.5-CTP, or a random peptide (RAN-Cy5.5), and euthanized at the indicated time points. (A) Peak transduction of heart tissue was seen at 15 min with steady decrease in fluorescence over time. Some capillary uptake was noted in the lungs with robust transduction in the liver as well at the kidney glomerular capillaries, the latter implying a renal mechanism of excretion. (B) Quantification of fluorescent intensity shows significantly increased heart uptake of CTP-Cy5.5 over RAN-Cy5.5. Scale bar = 500 &#181;m. This figure has been modified from Zahid et al.8. <a href="https://www.jove.com/files/ftp_upload/60895/60895fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide at JoVE.com

In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

Since the initial description of protein transduction domains, also known as cell penetrating peptides, over 25 years ago, there has been intense interest ...

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5.3K Views.  University of Pittsburgh School of Medicine. We describe protocols for assessing the degree of transduction by cell-penetrating peptides utilizing ex vivo imaging systems followed by paraffin embedding, sectioning, and confocal fluorescent microscopy using cardiac targeting peptide as an example. In our protocol, a single animal can be used to acquire both types of imaging assessment of the same organs, thereby cutting the number of animals needed for studies by half.

Video: In Vivo Imaging of Transduction Efficiencies of Cardiac Targeting Peptide

Targeted Expression of GFP in the Hair Follicle Using Ex Vivo Viral Transduction

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft during early anagen, the growth phase of the hair cycle, as well as pluripotent stem cells that play a role in hair follicle growth but have the potential to differentiate to non-follicle cells such as neurons. These properties of the hair follicle are discussed. The various cell types of the hair follicle are potential targets for gene therapy. Gene delivery system for the hair follicle using viral vectors or liposomes for gene targeting to the various cell types in the hair follicle and the results obtained are also discussed.

The hair follicle is a highly complex appendage of the skin containing a multiplicity of cell types. The follicle undergoes constant cycling through the life of the organism including growth and resorption with growth dependent on specific stem cells. The targeting of the follicle by genes and stem cells to change its properties, in particular, the nature of the hair shaft is, discussed. Hair follicle delivery systems are described, such as liposomes and viral vectors for gene therapy. The nature of the hair follicle stem cells is discussed, in particular, its pluripotency.

There are many cell types in the hair follicle, including hair matrix cells which form the hair shaft and stem cells which can initiate the hair shaft ...

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system is valuable for the pre-clinical in vivo evaluation of putative antitumor compounds.
The 4T1 cell line has been engineered to constitutively express the firefly luciferase gene (luc2). When mice carrying 4T1-luc2 tumors are injected with Luciferin the tumors emit a visual light signal that can be monitored using a sensitive optical imaging system like the IVIS Spectrum. The photon flux from the tumor is proportional to the number of light emitting cells and the signal can be measured to monitor tumor growth and development. IVIS is calibrated to enable absolute quantitation of the bioluminescent signal and longitudinal studies can be performed over many months and over several orders of signal magnitude without compromising the quantitative result.
Tumor growth can be monitored for several days by bioluminescence before the tumor size becomes palpable or measurable by traditional physical means. This rapid monitoring can provide insight into early events in tumor development or lead to shorter experimental procedures.
Tumor cell death and necrosis due to hypoxia or drug treatment is indicated early by a reduction in the bioluminescent signal. This cell death might not be accompanied by a reduction in tumor size as measured by physical means. The ability to see early events in tumor necrosis has significant impact on the selection and development of therapeutic agents.
Quantitative imaging of tumor growth using IVIS provides precise quantitation and accelerates the experimental process to generate results.

In vivo Bioluminescent Imaging of Mammary Tumors Using IVIS Spectrum

Mammary tumor cells expressing luciferase are implanted subcutaneously in mice and visualized using optical imaging to monitor tumor growth and development non-invasively in a longitudinal study.

4T1 mouse mammary tumor cells can be implanted sub-cutaneously in nu/nu mice to form palpable tumors in 15 to 20 days. This xenograft tumor model system ...

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

In vivo Imaging of Deep Cortical Layers using a Microprism

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size and have a reflective coating on the hypotenuse to allow internal reflection of excitation and emission light.  The microprism probe simultaneously images multiple cortical layers with a perspective typically seen only in slice preparations.  Images are collected with a large field-of-view (~900 &mu;m).  In addition, we provide details on the non-survival surgical procedure and microscope setup.  Representative results include images of layer V pyramidal neurons from Thy-1 YFP-H mice showing their apical dendrites extending through the superficial cortical layer and extending into tufts.  Resolution was sufficient to image dendritic spines near the soma of layer V neurons.  A tail-vein injection of fluorescent dye reveals the intricate network of blood vessels in the cortex.  Line-scanning of red blood cells (RBCs) flowing through the capillaries reveals RBC velocity and flux rates can be obtained. This novel microprism probe is an elegant, yet powerful new method of visualizing deep cellular structures and cortical function in vivo.

In vivo Imaging of Deep Cortical Layers using a Microprism

Right-angle microprisms inserted into the mouse neocortex allows for deep imaging of multiple cortical layers with a viewpoint typically found in slice. One-millimeter microprisms offer a wide field-of-view (~900 &mu;m) and spatial resolutions sufficient to resolve dendritic spines. We demonstrate layer V neuronal imaging and neocortical vascular imaging using microprisms.

We present a protocol for in vivo imaging of cortical tissue using a deep-brain imaging probe in the shape of a microprism.  Microprisms are 1-mm in size ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Intravital Microscopy of the Microcirculation in the Mouse Cremaster Muscle for the Analysis of Peripheral Stem Cell Migration

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to nonmarrow tissue have not been completely clarified. We describe here the technique of intravital microscopy applied to the mouse cremaster microcirculation for analysis of peripheral bone marrow stem cell migration in vivo. Intravital microscopy of the M. cremaster has been previously introduced in the field of inflammatory research for direct observation of leucocyte interaction with the vascular endothelium. Since sufficient peripheral stem and progenitor cell migration includes similar initial steps of rolling along and firm adhesion at the endothelial lining it is conceivable to apply the M. cremaster model for the observation and quantification of the interaction of intravasculary administered stem cells with the endothelium. As various chemical components can be selectively applied to the target tissue by simple superfusion techniques, it is possible to establish essential microenvironmental preconditions, for initial stem cell recruitment to take place in a living organism outside the bone marrow.

Intravital microscopy of the mouse M. cremaster microcirculation offers a unique and well-standardized in vivo model for the analysis of peripheral bone marrow stem cell migration.

In the era of intravascular cell application protocols in the context of regenerative cell therapy, the underlying mechanisms of stem cell migration to ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

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) ...