Name: immunofluorescent detection of two thymidine analogues (cldu and idu) in primary tissue
Uploaded: 2010-12-07T16:35:00
Duration: 10 min 55 s
Description: Watch this Scientific Journal Video about Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue at JoVE.com

Supported by the JDRF, the NIH (R01-DK081469), the Commonwealth of Pennsylvania (Center for Excellence in Regenerative Medicine grant 4100043362), the March of Dimes (Basil O'Connor Starter Scholar Research Award), and a University of Pennsylvania DERC pilot and feasibility grant (DK19525).

1. Labeling Tissues with CldU and IdU
<ol>
<li>Dissolve thymidine analogues (CldU or IdU) in water. Weigh 1mg/ mL of each chemical and add to separate glass bottles of distilled water. We typically prepare 1 liter of each at a time.</li>
	<li>Dissolve the chemicals on a stir plate or other form of agitator. CldU will dissolve in 10 min. of agitation at room temperature. IdU requires more than an hour of agitation at 37&deg;C in a shaker. Water sources can influence IdU solubility. If IdU remains unsuspended after overnight shaking, try a cleaner source of water. The solutions are stored at 4&deg;C in the dark.</li>
	<li>Administer the first thymidine containing solution (either CldU or IdU) to the mice for the desired labeling period. Do not allow mice to have access to unlabeled water during the period of label. When the designated labeling period is completed, begin a washout with unlabeled water for at least 24 hours (the thymidine containing analogues have serum half-lives of several hours). Administer the second thymidine containing solution (either CldU or IdU) to the mice for the desired labeling period. Refill water bottles regularly to prevent dehydration!</li>
	<li>Alternatively, mice can be labeled by sequential intraperitoneal injection of CdlU and then IdU. Prepare CldU or IdU at 10mg/ mL concentration. IdU may require drop-wise addition of concentrated sodium hydroxide solution followed by vigorous shaking to go into suspension. Do not add excess sodium hydroxide. Add a single drop of sodium hydroxide to a 10 mL solution of IdU followed by an hour of agitation at 37&deg;C in a shaker. If not in solution, repeat addition of sodium hydroxide. Inject 100mcg per g body weight into the intraperitoneal space. </li>
	<li>Control slides are essential for this protocol. As a result, we strongly suggest investigators carry out several variations of thymidine analogue labeling to ensure sensitivity and specificity. These should include 1. Mice that were only labeled with CldU; 2. Mice that were only labeled with IdU; 3. Mice that were sequentially labeled with CldU and then IdU; 4. Mice that were sequentially labeled in reverse order, first with IdU and then CldU; 5 Mice that are mock labeled with only water. When learning to label with CldU and IdU, investigators should always carry out the entire protocol with all 5 above sets of control slides from the tissue of interest.</li>
</ol>
2. Tissue Harvest, Fixation, and Slide Preparation
<ol>
	<li>Using appropriate anesthetic, perform lethal overdose and then sacrifice mouse via exsanguination. Remove fresh tissue and fix with 4% paraformalehyde at 4&deg;C for 24 hours. Transfer tissue to 1:4 formalin buffer and store at 4&deg;C for embedding. </li>
	<li>Alternatively, perform lethal overdose and then perfuse the mouse via the left cardiac ventricle with saline solution until blood is cleared from the body; then immediately perfuse with 30-50 cc of 4% paraformaldehyde. Remove the tissues of interest and store in 1:4 Formalin Buffer at 4&deg;C for embedding. </li>
	<li>After tissue dehydration and paraffin embedding, cut 5-micron tissue sections and place onto charged slides. Bake the slides at 65&deg;C for 16 hours to ensure adhesion of tissue to slide (essential with antigen retrieval steps below).</li>
</ol>
3. Dehydration, Permeabilization, and Antigen Retrieval
<ol>
	<li>Remove excess paraffin by immersing slides in two xylene baths for 5 min. each. </li>
	<li>Rehydrate the tissue by immersing slides in ethanol diluted with water: 100, 95, 90, 80 70 and 50%. Soak the slides for 3 min. at each concentration, starting with 100% and going towards 50% for the final immersion.</li>
	<li>Wash slides for 5 min. in 1x PBS.</li>
	<li>Permeabilize cells by immersing slides in 0.2% Triton-X solution for 5 min (made fresh every time).</li>
	<li>Prepare slides for microwave tissue antigen retrieval . Place the slides into a glass beaker with a sufficient volume 0.01M pH 6.0 sodium citrate buffer, so that the solution covers 5 cm above the top of the slides to account for evaporative loss.</li>
	<li>Microwave slides until the solution is boiling (3-10 min. at full power). Reduce power until solution is slowly boiling (10-80%) and continue for an additional 20 min. Check microwave periodically to ensure the solution remains at a slow boil. </li>
	<li>Place beaker containing slides on a bench-top and allow to gently cool to room temperature, (approximately 2 hours).</li>
	<li> Confirm that slides are at room temperature using a thermometer.</li>
	<li>Immerse slides in 1.5N HCL for 40 min. at room temperature. </li>
	<li> Wash slides twice in 1X PBS, 5 min. per wash.</li>
</ol>
4. Primary and Secondary Antibodies
<ol>
	<li>It is important that the slides remain very moist through the entire protocol. Dry tissue will have much more auto-fluorescence, making the resulting images unusable. To prevent dying, process one slide at a time. </li>
	<li>Circle each section on the slides with a liquid blocking (i.e., wax) pen. Place the slides back into the container of 1x PBS.</li>
	<li>Make a humid incubation chamber by placing wet paper towels laid flat on the bottom of an airtight plastic container.</li>
	<li>Make up blocking solution of 10% donkey serum diluted in 1x PBS. It is important to entirely cover each section with the solution. For tissue sections that are 1 x 1.5 centimeters, a volume of 50 micro-liters of solution per section is sufficient.</li>
	<li>Add the blocking solution to the slides. Remove slide from 1x PBS and eliminate excess liquid by gently tapping the slide edge on a stack of dry paper towels. Place slide in incubation chamber and add 50 micro-liters of 10% block solution to each section. When all the slides are completed close the container and incubate at room temperature for 1 hour.</li>
	<li>Prepare primary antibody dilution to detect IdU. Dilute mouse anti-BrdU at a 1:250 concentration in 5% donkey serum made in 1x PBS. Tap off block solution from slide and place back into the incubation chamber. Add the above primary antibody to each section and incubate overnight at 4&deg;C.</li>
	<li>Prepare high stringency wash (which minimizes binding by mouse anti-BrdU antisera to CldU). Make up fresh low salt TBST buffer (36mM Tris, 50mM NaCl, 0.5% tween-20; pH 8.0). You will need 40 mL of buffer per pair of slides. Add 40 mL of buffer to each 50 mL conical tube. Before adding slides, preheat the solution to 37&deg;C.</li>
	<li>Two at a time, remove slides from the incubation chamber, place them back-to-back (so that tissue samples face away from one another) and tap off excess primary antibody. Place the slides in the tubes filled with low salt TBST and cover. Agitate the tubes for 20 min. in a shaking bacterial culture incubator with the temperature at 37&deg;C, and the speed set to 225 rpm.</li>
	<li>Wash the slides two times in fresh 1X PBS. 5 min. per wash. </li>
	<li>Prepare the next primary antibody solution for detection of CldU and tissue antigen, if desired. Dilute rat anti-BrdU at a 1:250 concentration in 5% donkey serum made in 1x PBS. Dilute primary antisera against tissue antigen at working concentration into the primary antibody solution.</li>
	<li>Tap excess 1x PBS off of the slides, and place them in the incubation chamber. Add primary antibody to each section and incubate overnight at 4&deg;C. </li>
	<li>Wash the slides two times in fresh 1X PBS. 5 min. per wash.</li>
	<li>Prepare the secondary antibody solution. Dilute Cy2 donkey anti-rat to 1:250 and Cy5 donkey anti-mouse to 1:500 in a 1X PBS solution containing 5% donkey serum. Add Cy3 secondary to detect primary species of tissue antigen antisera. Also, dilute a 0.4mg/ mL stock solution of DAPI to 1:150 in secondary antibody solution.</li>
	<li>Tap excess 1X PBS off slides and place in the incubation chamber. Add secondary antibody solution to each section and incubate for 1 hour at room temperature. </li>
	<li>Wash the slides two times in fresh 1X PBS. 5 min. per wash.</li>
	<li>Tap excess 1X PBS off of the slides and adhere cover glass with Prolong Gold anti-fade mounting media (DO NOT use Vectashield with dyanine dyes!). Press on cover slips to eliminate air bubbles. Store at 4&deg;C.</li>
</ol>
5. Imaging and Analysis
<ol>
	<li>Image the tissue of interest with a fluorescent microscope equipped with a high energy light source, plan-apochromatic objectives, and a high efficiency microscope camera (e.g. Hamamatsu Orca ER). Capture images in the AMCA (DAPI), Cy2 (CldU), Cy3 (Tissue antigen), and Cy5 (IdU) channels. Merge images to create a single multi-layer image file.</li>
	<li>If performed correctly, DAPI stained nuclei will be homogeneously bright. Tissue antigen (e.g. insulin) should reveal cells of interest. If staining with DAPI or tissue-antigen is weak or inconsistent, immerse slide in 1X PBS overnight to remove cover slip and repeat application of secondary antisera.</li>
	<li>Focus on tissue antigen in Cy3. Check CldU and IdU staining in Cy2 and Cy5 channels, toggling between channels. Look for potential cross-reactivity between markers, as indicated by excessive IdU and CldU co-positivity. Look for tissues that contain only CldU or IdU. Lack of CldU or IdU positive nuclei may indicate a technical problem with labeling or staining. In this case, look for a highly replicative tissue (e.g. lymph node).</li>
	<li>Analyze images. Display layers containing DAPI and tissue antigen. Using a white board marker in the dominant hand and a cell counter in the non-dominant hand, mark each nucleus with a contrasting mark with a dry erase marker (a.k.a. white board marker). Count DAPI positive cells within tissue of interest. Record total cell count. Use a dry eraser to remove marks. Display layers containing DAPI, tissue antigen, and CldU. Count CldU positive cells within tissue of interest. Record CldU cell count but do not erase marks. Change display to visualize layers containing tissue antigen, CldU, and IdU. Count CldU IdU double positive cells. Record value, erase all marks, and display layers containing DAPI, tissue antigen, and IdU. Count the total number of IdU positive cells within the tissue of interest. Record IdU cell count.</li>
</ol>
6. Representative Results 
We sequentially labeled 6 week old female mice with CldU for one week and then IdU for two weeks, followed by immediate sacrifice. Pancreata were stained to detect CldU, IdU, and Insulin (a tissue antigen), as well as DAPI. Islets were uniformly stained with insulin. CldU stained nuclei were distinct from IdU stained nuclei (Figure 2). Many nuclei outside of the islet were CldU IdU co-positive (Figure 2, bottom right, white arrows). A single insulin positive cell had nuclear staining for both CldU and IdU (Figure 2, bottom right, magenta arrow). 
Mice. All experiments with mice were performed according to the guidelines of the IACUC committee of the Children's Hospital of Philadelphia. Female B6.129 F1 wild-type mice were purchased from Taconic (Germantown New York), housed at the laboratory animal facility at The Children's Hospital of Philadelphia, and fed Mouse Diet 5015 from PMI Nutrition International (Richmond. Indiana). 
Labeling strategy. By labeling the first cell division event with CldU (marked green in this scheme) and the second cell division with IdU (marked red in this scheme), sequential cell division results in co-labeled cells with both CldU and IdU (green/red). 
Representative results of thymidine labeling of pancreatic cell turnover within mice. Immunofluorescent image of a mouse pancreas with islet of langerhans in the center. Mouse was infused with CldU for 1 week and then IdU for 2 weeks in the drinking water prior to immediate sacrifice. Pancreas sample was processed as described in protocol. 40x objective images, with distinct layers displayed suitable for counting. Merged images contain DAPI (white) CldU (green), IdU (red), Insulin (yellow). (upper, left) DAPI and insulin. (upper, right) CldU and Insulin. (lower, left) IdU and Insulin. (lower, right) CldU, IdU, and Insulin. Scale bar: 100&mu;m.
<img src="/files/ftp_upload/2166/2166fig1.jpg" alt="Figure 1" /> Figure 1. Labeling strategy. By labeling the first cell division event with CldU (marked green in this scheme) and the second cell division with IdU (marked red in this scheme), sequential cell division results in co-labeled cells with both CldU and IdU (green/red).
<img src="/files/ftp_upload/2166/2166fig2.jpg" alt="Figure 2" /> Figure 2. Representative results of thymidine labeling of pancreatic cell turnover within mice. Immunofluorescent image of a mouse pancreas with islet of langerhans in the center. Mouse was infused with CldU for 1 week and then IdU for 2 weeks in the drinking water prior to immediate sacrifice. Pancreas sample was processed as described in protocol. 40x objective images, with distinct layers displayed suitable for counting. Merged images contain DAPI (white) CldU (green), IdU (red), Insulin (yellow). (upper, left) DAPI and insulin. (upper, right) CldU and Insulin. (lower, left) IdU and Insulin. (lower, right) CldU, IdU, and Insulin. Scale bar: 100&mu;m.

<ol>
	<li>Teta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+regeneration+of+adult+Beta+cells+does+not+involve+specialized+progenitors.">Growth and regeneration of adult Beta cells does not involve specialized progenitors.</a> Dev Cell. 12 (5), 817-817 (2007).</li><li>Kiel, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+stem+cells+do+not+asymmetrically+segregate+chromosomes+or+retain+BrdU.">Haematopoietic stem cells do not asymmetrically segregate chromosomes or retain BrdU.</a> Nature. 449 (7159), 238-238 (2007).</li><li>Bersell, K., Arab, S., Haring, B., Kuhn, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuregulin1/ErbB4+signaling+induces+cardiomyocyte+proliferation+and+repair+of+heart+injury.">Neuregulin1/ErbB4 signaling induces cardiomyocyte proliferation and repair of heart injury.</a> Cell. 138, 257-257 (2009).</li><li>Rankin, M. M., Kushner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+Beta+Cell+Proliferation+Is+Severely+Restricted+with+Advanced+Age.">Adaptive Beta Cell Proliferation Is Severely Restricted with Advanced Age.</a> Diabetes. , (2009).</li><li>He, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyclin+D2+Protein+Stability+Is+Regulated+in+Pancreatic+Beta+Cells.">Cyclin D2 Protein Stability Is Regulated in Pancreatic Beta Cells.</a> Mol Endocrinol. , (2009).</li><li>Kushner, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beta-cell+growth:+an+unusual+paradigm+of+organogenesis+that+is+cyclin+D2/Cdk4+dependent.">Beta-cell growth: an unusual paradigm of organogenesis that is cyclin D2/Cdk4 dependent.</a> Cell Cycle. 5 (3), 234-234 (2006).</li><li>Teta, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Very+slow+turnover+of+beta-cells+in+aged+adult+mice.">Very slow turnover of beta-cells in aged adult mice.</a> Diabetes. 54 (9), 2557-2557 (2005).</li><li>Wilson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hematopoietic+stem+cells+reversibly+switch+from+dormancy+to+self-renewal+during+homeostasis+and+repair.">Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair.</a> Cell. 135 (6), 1118-1118 (2008).</li><li>Van Nest, G., Raman, R. K., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+dexamethasone+and+5-bromodeoxyuridine+on+protein+synthesis+and+secretion+during+in+vitro+pancreatic+development.">Effects of dexamethasone and 5-bromodeoxyuridine on protein synthesis and secretion during in vitro pancreatic development.</a> Dev Biol. 98 (2), 295-295 (1983).</li><li>Githens, S., Pictet, R., Phelps, P., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-bromodeoxyuridine+may+alter+the+differentiative+program+of+the+embryonic+pancreas.">5-bromodeoxyuridine may alter the differentiative program of the embryonic pancreas.</a> J Cell Biol. 71 (2), 341-341 (1976).</li><li>Walther, B. T., Pictet, R. L., David, J. D., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+the+mechanism+of+5-bromodeoxyuridine+inhibition+of+exocrine+pancreas+differentiation.">On the mechanism of 5-bromodeoxyuridine inhibition of exocrine pancreas differentiation.</a> J Cell Biol. 249 (6), 1953-1953 (1974).</li><li>Chen, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+pancreatic+islet+cell+proliferation+by+heavy+water+labeling.">Measurement of pancreatic islet cell proliferation by heavy water labeling.</a> Am J Physiol Endocrinol Metab. 293 (5), E1459-E1459 (2007).</li><li>Salic, A., Mitchison, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proc Natl Acad Sci U S A. 105 (7), 2415-2415 (2008).</li><li>Best, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Click+chemistry+and+bioorthogonal+reactions:+unprecedented+selectivity+in+the+labeling+of+biological+molecules.">Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules.</a> Biochemistry. 48 (28), 6571-6571 (2009).</li><li>Buck, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+S-phase+cell+cycle+progression+using+5-ethynyl-2'-deoxyuridine+incorporation+with+click+chemistry,+an+alternative+to+using+5-bromo-2'-deoxyuridine+antibodies.">Detection of S-phase cell cycle progression using 5-ethynyl-2'-deoxyuridine incorporation with click chemistry, an alternative to using 5-bromo-2'-deoxyuridine antibodies.</a> Biotechniques. 44 (7), 927-927 (2008).</li><li>Huising, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRFR1+is+expressed+on+pancreatic+beta+cells,+promotes+beta+cell+proliferation,+and+potentiates+insulin+secretion+in+a+glucose-dependent+manner.">CRFR1 is expressed on pancreatic beta cells, promotes beta cell proliferation, and potentiates insulin secretion in a glucose-dependent manner.</a> Proc Natl Acad Sci U S A. 107 (2), 912-912 (2008).</li><li>Reimann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucose+sensing+in+L+cells:+a+primary+cell+study.">Glucose sensing in L cells: a primary cell study.</a> Cell Metab. 8 (6), 532-532 (2008).</li><li>Doupe, D. P., Klein, A. M., Simons, B. D., Jones, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ordered+architecture+of+murine+ear+epidermis+is+maintained+by+progenitor+cells+with+random+fate.">The ordered architecture of murine ear epidermis is maintained by progenitor cells with random fate.</a> Dev Cell. 18 (2), 317-317 (2010).</li></ol>

All experiments with mice were performed in the animal facility at The Children's Hospital of Philadelphia according to the guidelines of the Institutional Animal Care and Use Committee (IACUC).

Our approach to thymidine-based approach to labeling cell turnover has many potential applications in biomedical research. To date, we have concentrated on the pancreas, but we have also applied this strategy to examine cell turnover of skin, gut, liver, adrenal, kidney, testis, ovary, thyroid, lymph node, hematopoiesis, and brain 1. Because cell turnover varies from tissue to tissue, labeling strategies should be customized to obtain the most compelling information from the organ of interest. Morrison and colleagues used CldU and IdU for 1 day each to label hematopoiesis 2. Kuhn and colleagues use CldU and IdU for 4 days each to detect cell division in regenerating myocardium 3. In contrast, we have used CldU and IdU for up to 9 months total to label basal cell turnover within pancreatic islets, a tissue with minimal cell turnover as the animal ages 1,4-6. The most obvious potential application of our labeling strategy is for studies to define cell turnover within tissue homeostasis. But our technique has several other potential applications. For instance, we also recently applied this technique to identify oncogenic transformation within tissues, where focal areas of increased replication can be readily identified by increased incorporation of CldU or IdU 5. Morrison and colleagues used CldU and IdU for 1 day each to test for label retaining behavior of hematopoietic stem cells 2. We have also applied sequential thymidine analogue labeling to determine if transit-amplifying cells contribute to growth or renewal of tissues 1. In this application sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.
Thymidine analogues should not be used without caution. Synthetic thymidine analogues have potential toxicities to dividing cells, and their use could theoretically impair cell turnover in growing animals. Thymidine analogues have been used as radiosensitizing agents for cancer patients, and may slow cell turnover. Thus we urge investigators to cautiously investigate for potential toxicities in their tissue of interest. For example, Trumpp and colleagues find that systemic BrdU can force hematopoietic stem cells to enter cell cycle 7. For instance, Rutter and colleagues observed that high dose thymidine analogues are toxic to developing pancreas 8. More recently Hellerstein and colleagues at KineMed, Inc found that BrdU administration slows intra-islet proliferation by 16-25% using a proprietary heavy water labeling technique 9. The whole islet preparations used by Hellerstein and colleagues contained many other endocrine and non-endocrine cell types, which could comprise as much as 50% of total islet preparations. However, we find that prolonged infusion of BrdU does not influence beta-cell proliferation in young adult mice, as measured by ki67 expression 4. Similarly, long-term administration of CldU and IdU does not seem to impair beta cell mass expansion 1. Moreover, beta cell proliferation rates are equivalent in between mice labeled with BrdU for long periods of time and mice that are only labeled for a few hours (rates are normalized to equivalent time periods). Together, these results suggest that mice can safely tolerate long-term low-dose administration of thymidine analogues. Still, we cannot rule out potential toxicity by thymidine analogues in pancreatic beta cell growth. As a result, we continue to perform controls when labeling mice with thymidine analogues, and urge other investigators to do so as well.
Our technique of sequential thymidine analogue labeling is not without pitfalls and technical challenges. As a result, control slides are especially important. We have generated control slides that consist of various tissues from 1.) Mice that were only labeled with CldU; 2.) Mice that were only labeled with IdU; 3.) Mice that were sequentially labeled with CldU and then IdU; 4.) Mice that were sequentially labeled in reverse order, first with IdU and then CldU; 5.) Mice that are mock labeled with only water. When learning to label with CldU and IdU, investigators should always carry out the entire protocol with all 5 above sets of slides from the tissue of interest.
Slight cross reactivity in between CldU and IdU is an unfortunate but unavoidable downside to labeling with both thymidine analogues. The CldU and IdU technique is based on differences in affinities in between antisera that were originally derived against BrdU in different species (mouse and rat). Our protocol, while cumbersome, is designed to minimize such cross reactivity. Consequently, we urge caution amongst investigators who consider skipping step(s) of the protocol. In particular, we have encountered problems with secondary antisera that are cross reactive in between mouse and rat. This can be minimized by the use of Jackson antisera that are fully cross-adsorbed in between mouse and rat.
We occasionally fail to adequately detect thymidine incorporation. In such instances it is critically important to use positive control slides to determine which reagent or step was not working. Difficulties are typically due to bad aliquots of antisera, dry slides, or inadequate nuclear permeabilization. We have failed to successfully label developing embryos with IdU. Thus, not all tissues may be permeable to IdU.
Alternatives to CldU and IdU for double thymidine analogue labeling are on the horizon. EdU (5-ethinyl-2 deoxyuridine) can be a substrate for copper catalyzed click chemistry detection 10,11. EdU detection methods do not require separation of DNA strands, and are thus highly amenable to analysis by flow cytometry 12. EdU is compatible but not cross reactive with BrdU 10. EdU has been used to quantify cell turnover of various mouse tissues including pancreatic beta cells 13, intestinal L cells 14 and Epidermis 15. EdU is fairly expensive at the moment ($1000 US per 500mg). Still, Edu detection appears to be much more sensitive than BrdU detection 10. Thus, it might be economically feasible to combine EdU and BrdU instead of CldU and IdU. This method might also allow detection of three different rounds of cell division in mice triply labeled for EdU, CldU, and IdU.
In summary, our technique of sequential thymidine analogue labeling is a novel approach to detecting cell turnover. We hope our approach enables other scientists to more accurately quantify cell division, and expect that it will open new paradigms in tissue homeostasis.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>5-chloro-2-deoxyuridine (CldU)</td>
<td>&nbsp;</td>
<td>MP Biomedical</td>
<td>0210547891</td>
<td>&nbsp;</td>
<tr>
<td>5-Iodo-2-deoxyuridine (IdU)</td>
<td>&nbsp;</td>
<td>MP Biomedical</td>
<td>0210035701</td>
<td>&nbsp;</td>
<tr>
<td>4% Paraformaldehyde</td>
<td>&nbsp;</td>
<td>USB</td>
<td>19943</td>
<td>&nbsp;</td>
<tr>
<td>10% Buffered Formalin</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>23-427-098</td>
<td>&nbsp;</td>
<tr>
<td>XYLENES</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>EM-XX0060-4</td>
<td>&nbsp;</td>
<tr>
<td>Ethanol, Anhydrous (Histological)</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>A405P-4</td>
<td>&nbsp;</td>
<tr>
<td>PBS, 10X Powder Concentrate</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP665-1</td>
<td>&nbsp;</td>
<tr>
<td>Triton &#x2013;X-100</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>BP151-500</td>
<td>&nbsp;</td>
<tr>
<td>Hydrochloric Acid</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>BDH3028-2.5LG</td>
<td>&nbsp;</td>
<tr>
<td>Normal Donkey Serum</td>
<td>&nbsp;</td>
<td>Jackson ImmunoResearch</td>
<td>017-000-121</td>
<td>&nbsp;</td>
<tr>
<td>Mouse Anti BrdU</td>
<td>&nbsp;</td>
<td>BD Pharmingen</td>
<td>347580</td>
<td>&nbsp;</td>
<tr>
<td>Rat mab anti BrdU</td>
<td>&nbsp;</td>
<td>Accurate Chemical</td>
<td>OBT0030</td>
<td>&nbsp;</td>
<tr>
<td>Cy2 donkey anti-rat</td>
<td>&nbsp;</td>
<td>Jackson Immuno</td>
<td>712-225-153</td>
<td>Fully adsorbed secondaries from Jackson (minimal cross reactivity).</td>
</tr>
<tr>
<td>Cy5 donkey anti-mouse</td>
<td>&nbsp;</td>
<td>Jackson immuno</td>
<td>715-175-151</td>
<td>See above</td>
</tr>
<tr>
<td>Glass Cover Slips</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>C9056-1CS</td>
<td>&nbsp;</td>
<tr>
<td>YFP Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49003 ET EYFP</td>
<td>Reduces auto-fluorescence in Cy2 channel</td>
</tr>
<tr>
<td>Cy3 Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49004 ET Cy3</td>
<td>&nbsp;</td>
<tr>
<td>Cy5 Filter</td>
<td>&nbsp;</td>
<td>Chroma</td>
<td>49006 ET Cy5</td>
<td>&nbsp;</td>
<tr>
<td>ORCA ER Microscope Camera</td>
<td>&nbsp;</td>
<td>Hamamatsu</td>
<td>C4742-95-12ER</td>
<td>Excellent detection of fluorophores</td>
</tr>
<tr>
<td>ProLong Gold antifade reagent</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>P36930</td>
<td>Do not use Vectashield (toxic to cyanine dyes).</td>
</tr>		</tbody>
		</table>
		</div>

immunofluorescent detection of two thymidine analogues (cldu and idu) in primary tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are analyzed. Established methods to detect cell division include direct visualization by continuous microscopy in cell culture, dilution of vital dyes such as carboxy&#64258;uorescein di-aetate succinimidyl ester (CFSE), immuno-detection of mitogenic antigens such as ki67 or PCNA, and thymidine analogues. Thymidine analogues can be detected by a variety of methods including radio-detection for tritiated thymidine, immuno-detection for bromo-deoxyuridine (BrdU), chloro-deoxyuridine (CldU) and iodo-deoxyuridine (IdU), and chemical detection for ethinyl-deoxyuridine (EdU). We have derived a strategy to detect sequential incorporation of different thymidine analogues (CldU and IdU) into tissues of adult mice. Our method allows investigators to accurately quantify two successive rounds of cell division. By optimizing immunostaining protocols our approach can detect very low dose thymidine analogues administered via the drinking water, safe to administer to mice for prolonged periods of time. Consequently, our technique can be used to detect cell turnover in very long-lived tissues. Optimal immunofluoresent staining results can be achieved in multiple tissue types, including pancreas, skin, gut, liver, adrenal, testis, ovary, thyroid, lymph node, and brain. We have also applied this technique to identify oncogenic transformation within tissues. We have further applied this technique to determine if transit-amplifying cells contribute to growth or renewal of tissues. In this sense, sequential administration of thymidine analogues represents a novel approach for studying the origins and survival of cells involved in tissue homeostasis.

Watch this Scientific Journal Video about Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue at JoVE.com

Immunofluorescent Detection of Two Thymidine Analogues CldU and IdU in Primary Tissue

We have derived a strategy to detect sequential incorporation of thymidine analogues (CldU and IdU) into tissues of adult mice to quantify two successive rounds of cell division. This strategy is useful to detect cell turnover of long-lived tissues, oncogenic transformation, or transit-amplifying cells.

Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue

Accurate measurement of cell division is a fundamental challenge in experimental biology that becomes increasingly complex when slowly dividing cells are ...

immunofluorescent-detection-two-thymidine-analogues-cldu-idu-primary

Research

JoVE Journal

Biology

Isolation and Primary Culture of Rat Hepatic Cells

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology and other related subjects. The method based on two-step collagenase perfusion for isolation of intact hepatocytes was first introduced by Berry and Friend in 1969 1 and, since then, has undergone many modifications. The most commonly used technique was described by Seglenin 1976 2. Essentially, hepatocytes are dissociated from anesthetized adult rats by a non-recirculating collagenase perfusion through the portal vein. The isolated cells are then filtered through a 100 &mu;m pore size mesh nylon filter, and cultured onto plates. After 4-hour culture, the medium is replaced with serum-containing or serum-free medium, e.g. HepatoZYME-SFM, for additional time to culture. These procedures require surgical and sterile culture steps that can be better demonstrated by video than by text. Here, we document the detailed steps for these procedures by both video and written protocol, which allow consistently in the generation of viable hepatocytes in large numbers.

Primary hepatocytes provide a valuable tool to evaluate biochemical, molecular, and metabolic functions in a physiologically relevant experimental system. We describe a reliable protocol for rat in situ liver perfusion, which consistently generates viable hepatocytes up to 1.0 &times; 108 cells per preparation with cell viability between 88 ~ 96%.

Primary hepatocyte culture is a valuable tool that has been extensively used in basic research of liver function, disease, pathophysiology, pharmacology ...

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

Improved Swiss-rolling Technique for Intestinal Tissue Preparation for Immunohistochemical and Immunofluorescent Analyses

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current literature describes several different methodological approaches to obtain images of intestinal tissues for data validation. In this paper, we delineate a common protocol relating to the derivation and processing of mouse intestinal tissues. Proper fixation of intestinal tissues and Swiss-roll techniques that enhance intestinal epithelial morphology are discussed. Postresection processing and reorientation of embedded intestinal tissues are critical in obtaining paraffin-embedded blocks that display intact intestinal structural features after sectioning. The Swiss-rolling technique helps in histological assessment of the complete intestinal or colonic sections examined. An ability to differentiate intestinal structural features can be vital in quantitative measurements of intestinal inflammation and tumorigenesis along the entire length. Finally, paraffin-embedded sections are ideal for robust processing using both immunohistochemical and immunofluorescent detection methods. Nonfluorescent immunohistochemical sections provide a vibrant image of the tissue detailing different cellular structural features but do not provide flexibility for intracellular co-localization experiments. Multiple fluorescent channels can be appropriately utilized with immunofluorescent detection for co-localization experiments, lending support to mechanistic studies.

Accurate identification and location of epithelial cells along the intestinal mucosal lining are essential to define different cell lineages. Proper imaging of intestinal tissues is crucial for identification of protein expression patterns with maximum resolution. This study aims to delineate the optimal methods and conditions for processing mouse intestinal tissues.

Understanding the role of factors that regulate intestinal epithelial homeostasis and response to injury and regeneration is important. The current ...

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal muscle development and regeneration, and the autophagic process has been described in several muscular pathologies and age-related muscle disorders. A recently described block of the autophagic process that correlates with the functional exhaustion of satellite cells during muscle repair supports the notion that active autophagy is coupled with productive muscle regeneration. These data uncover the crucial role of autophagy in satellite cell activation during muscle regeneration in both normal and pathological conditions, such as muscular dystrophies. Here, we provide a protocol to monitor the autophagic process in the adult Muscle Stem Cell (MuSC) compartment during muscle regenerative conditions. This protocol describes the setup methodology to perform in situ immunofluorescence imaging of LC3, an autophagy marker, and MyoD, a myogenic lineage marker, in muscle tissue sections from control and injured mice. The methodology reported allows for monitoring the autophagic process in one specific cell compartment, the MuSC compartment, which plays a central role in orchestrating muscle regeneration.

In Situ Immunofluorescent Staining of Autophagy in Muscle Stem Cells

Active autophagy is associated with productive muscle regeneration, which is essential for Muscle Stem Cell (MuSC) activation. Here, we provide a protocol for the in situ detection of LC3, an autophagy marker in MyoD-positive MuSCs of muscle tissue sections from control and injured mice.

Increasing evidence points to autophagy as a crucial regulatory process to preserve tissue homeostasis. It is known that autophagy is involved in skeletal ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Microbiota Analysis Using Two-step PCR and Next-generation 16S rRNA Gene Sequencing

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the immune system. Perturbation of the healthy gut microbiome has been suggested to play a role in the development of inflammatory diseases, including multiple sclerosis (MS). Environmental and genetic factors can influence the composition of the microbiome; therefore, identification of microbial communities linked with a disease phenotype has become the first step towards defining the microbiome&#8217;s role in health and disease. Use of 16S rRNA metagenomic sequencing for profiling bacterial community has helped in advancing microbiome research. Despite its wide use, there is no uniform protocol for 16S rRNA-based taxonomic profiling analysis. Another limitation is the low resolution of taxonomic assignment due to technical difficulties such as smaller sequencing reads, as well as use of only forward (R1) reads in the final analysis due to low quality of reverse (R2) reads. There is need for a simplified method with high resolution to characterize bacterial diversity in a given biospecimen. Advancements in sequencing technology with the ability to sequence longer reads at high resolution have helped to overcome some of these challenges. Present sequencing technology combined with a publicly available metagenomic analysis pipeline such as R-based Divisive Amplicon Denoising Algorithm-2 (DADA2) has helped advance microbial profiling at high resolution, as DADA2 can assign sequence at the genus and species levels. Described here is a guide for performing bacterial profiling using two-step amplification of the V3-V4 region of the 16S rRNA gene, followed by analysis using freely available analysis tools (i.e., DADA2, Phyloseq, and METAGENassist). It is believed that this simple and complete workflow will serve as an excellent tool for researchers interested in performing microbiome profiling studies.

Described here is a simplified standard operating procedure for microbiome profiling using 16S rRNA metagenomic sequencing and analysis using freely available tools. This protocol will help researchers who are new to the microbiome field as well as those requiring updates on methods to achieve bacterial profiling at a higher resolution.

The human gut is colonized by trillions of bacteria that support physiologic functions such as food metabolism, energy harvesting, and regulation of the ...

Apoptosis Induction and Detection in a Primary Culture of Sea Cucumber Intestinal Cells

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological substances or characterization of specific biological activities. However, due to the lack of universally applicable cell culture media and protocols, well described cell culture methods for marine organisms are still limited. Meanwhile, the commonly occurring microbial contamination and polytropic properties of marine invertebrate cells further impede the establishment of an effective cell culture strategy for marine invertebrates. Here, we describe an easy-to-handle method for culturing intestinal cells from sea cucumber Apostichopus japonicus; additionally, we provide an example of in vitro apoptosis induction and detection in primary cultured intestinal cells. Moreover, this experiment provides details about the appropriate culture medium and cell collection method. The described protocol is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea, and it can provide sufficient cells for multiple in vitro experimental applications. This technique would enable researchers to efficiently manipulate primary cell cultures from marine invertebrates and to facilitate the functional evaluation of targeted biological materials on cells.

This protocol provides an easy-to-handle method to culture the intestinal cells from sea cucumber Apostichopus japonicus and is compatible with a variety of widely available tissue samples from marine organisms including Echinodermata, Mollusca, and Crustacea.

Primary cultured cells are used in a variety of scientific disciplines as exceptionally important tools for the functional evaluation of biological ...

Simple Detection of Primary Cilia by Immunofluorescence

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to mitosis. Primary cilia can be visualized with highly sophisticated methods, including transmission electron microscopy, 3D imaging, or using software for the automatic detection of primary cilia. However, immunofluorescent staining of primary cilia is needed to perform these methods. This publication describes a protocol for the easy detection of primary cilia in vitro by staining acetylated alpha tubulin (axoneme) and gamma tubulin (basal body). This immunofluorescent staining protocol is relatively simple and results in high-quality images. The present protocol describes how four cell lines (C2C12, MEF, NHLF, and skin fibroblasts) expressing primary cilia were fixed, immunostained, and imaged with a fluorescent or confocal microscope.

Primary cilia are extracellular structures associated with the centriole. Primary cilia detection by immunofluorescent staining is a relatively simple procedure that results in extremely high-quality images. In this protocol, fibroblasts expressing primary cilia were fixed, immunostained, and imaged in a fluorescent or confocal microscope.

Primary cilia are dynamically regulated during cell cycle progression, specifically during the G0/G1 phases of the cell cycle, being resorbed prior to ...

Quantitative Proteomics Workflow using Multiple Reaction Monitoring Based Detection of Proteins from Human Brain Tissue

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related disorders continue to be a major contributor of deaths around the world, necessitating the need for even greater understanding of their pathobiology. Traditional antibody-based techniques like western blotting or immunohistochemistry suffer from being low-throughput besides being labor-intensive and qualitative or semi-quantitative. Even conventional mass spectrometry-based shotgun approaches fail to provide conclusive evidence to support a certain hypothesis. Targeted proteomics approaches are largely hypothesis driven and differ from the conventional shotgun proteomics approaches that have been long in use. Multiple reaction monitoring is one such targeted approach that requires the use of a special mass spectrometer called the tandem quadrupole mass spectrometer or triple quadrupole mass spectrometer. In the current study, we have systematically highlighted the major steps involved in performing a successful tandem quadrupole mass spectrometry-based proteomics workflow using human brain tissue with an aim to introduce this workflow to a broader research community.

The protocol aims to introduce the use of a triple quadrupole mass spectrometer for Multiple Reaction Monitoring (MRM) of proteins from clinical samples. We have provided a systematic workflow starting from sample preparation to data analysis for clinical samples with all the necessary precautions to be taken.

The proteomic analysis of the human brain tissue over the last decade has greatly enhanced our understanding of the brain. However, brain related ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...