Division of Endocrinology and Diabetes, Children’s Hospital of Philadelphia, Institute of Diabetes Obesity and Metabolism, Institute for Regenerative Medicine, Department of Pediatrics, University of Pennsylvania-School of Medicine
Tuttle, A. H., Rankin, M. M., Teta, M., Sartori, D. J., Stein, G. M., Kim, G. J., et al. Immunofluorescent Detection of Two Thymidine Analogues (CldU and IdU) in Primary Tissue. J. Vis. Exp. (46), e2166, doi:10.3791/2166 (2010).
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 carboxyﬂuorescein 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.
1. Labeling Tissues with CldU and IdU
2. Tissue Harvest, Fixation, and Slide Preparation
3. Dehydration, Permeabilization, and Antigen Retrieval
4. Primary and Secondary Antibodies
5. Imaging and Analysis
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: 100μm.
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).
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: 100μm.
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.
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).
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).
|5-chloro-2-deoxyuridine (CldU)||MP Biomedicals||0210547891|
|5-Iodo-2-deoxyuridine (IdU)||MP Biomedicals||0210035701|
|4% Paraformaldehyde||USB Corp., Affymetrix||19943|
|10% Buffered Formalin||Fisher Scientific||23-427-098|
|Ethanol, Anhydrous (Histological)||Fisher Scientific||A405P-4|
|PBS, 10X Powder Concentrate||Fisher Scientific||BP665-1|
|Triton €“X-100||Fisher Scientific||BP151-500|
|Hydrochloric Acid||VWR international||BDH3028-2.5LG|
|Normal Donkey Serum||Jackson ImmunoResearch||017-000-121|
|Mouse Anti BrdU||BD Biosciences||347580|
|Rat mab anti BrdU||Accurate Chemical & Scientific Corporation||OBT0030|
|Cy2 donkey anti-rat||Jackson ImmunoResearch||712-225-153||Fully adsorbed secondaries from Jackson (minimal cross reactivity).|
|Cy5 donkey anti-mouse||Jackson ImmunoResearch||715-175-151||See above|
|Glass Cover Slips||Sigma-Aldrich||C9056-1CS|
|YFP Filter||Chroma Technology Corp.||49003 ET EYFP||Reduces auto-fluorescence in Cy2 channel|
|Cy3 Filter||Chroma Technology Corp.||49004 ET Cy3|
|Cy5 Filter||Chroma Technology Corp.||49006 ET Cy5|
|ORCA ER Microscope Camera||Hamamatsu Corp.||C4742-95-12ER||Excellent detection of fluorophores|
|ProLong Gold antifade reagent||Invitrogen||P36930||Do not use Vectashield (toxic to cyanine dyes).|