Technically these two antibodies have their limitations Both

Technically, these two GDC0941 Supplier have their limitations. Both are better suited for immunofluorescent analysis in frozen tissue sections than in formalin-fixed paraffin-embedded tissues. In addition, anti-Lrig1-VU did not work for immunoprecipitation under our experimental conditions. FACS analysis is feasible using both antibodies, but the gating strategy used to separate Lrig1+ cells from background with the Lrig1-R&D antibody is not ideal. Finally, the two antibodies are not conducive to biochemical experiments when used simultaneously. We were unable to perform FACS analysis by sequential co-staining with both antibodies, suggesting that they may compete for Lrig1 binding in vitro. It should be noted that we observe non-specific staining in the upper crypt with anti-Lrig1-VU. This effect is enhanced upon simultaneous staining with anti-Lrig1-R&D, which also displays similar non-specific staining under these conditions (Fig. 1C-C‴). This study describes a useful new tool for the study of Lrig1: the Lrig1-Apple reporter mouse. Other intestinal stem cell reporter mice, such as Lgr5 (Barker et al., 2007; Breault et al., 2008) and mTert (Breault et al., 2008) express green fluorescent protein (GFP). The Lrig1-Apple RFP reporter is therefore perfectly compatible with such GFP reporters to compare multiple stem cell populations in the same tissue. As Lrig1 is broadly expressed in many tissues (Nilsson et al., 2003), the Lrig1-Apple mouse will be a useful tool to examine the role of Lrig1 outside the intestine. It is important to note that in this model, RFP serves as a readout of Lrig1 transcriptional activity only and may not accurately reflect the true regulated Lrig1 transcriptional unit or protein status in real time. Although profiling of the entire Lrig1+ population is still lacking (but feasible using anti-Lrig1-R&D), RNA-Seq analysis demonstrated that the anti-Lrig1-VU+ cell profile is characteristic of a stem cell population (Powell et al., 2012). Immunofluorescent analysis using anti-Lrig1-VU demonstrates that the position of this subpopulation in the colonic crypt varies; it should be emphasized that the anti-Lrig1-VU+ cell position is not restricted to the crypt base columnar cell zone or the +4 position, but varies, most often occupying positions 2–5 (quantified in Powell et al., 2012). In summary, we believe this study will be of interest to the intestinal stem cell field. There has been a reluctance to accept Lrig1 as an intestinal stem cell marker because of the discrepancies between the two Lrig1 studies published in 2012 (Powell et al., 2012; Wong et al., 2012). Here, we hope to have clarified differences between two Lrig1 antibodies and the respective Lrig1+ populations they recognize. We suggest the following usage for study of mouse Lrig1: to study all Lrig1+ and Lrig1+ cells in the intestinal crypt, we recommend using anti-Lrig1-R&D; when studying the Lgr5-/Lrig1+ stem cell subpopulation, we recommend using anti-Lrig1-VU. In addition, real time comparison of Lrig1+ cells with other stem cell populations expressing GFP reporters is now possible with the use of Lrig1-Apple reporter mice in conjunction with anti-Lrig1-VU. The following are the supplementary data related to this article.
Conflict of interest
Acknowledgments The authors thank the Vanderbilt University Mouse/Embryonic Stem Cell, Cell Imaging, and Flow Cytometry Shared Resources for aid in generating the Lrig1-Apple mouse, imaging, and flow cytometry resources, respectively. The authors also thank Jim Higginbotham, Ph.D. for flow cytometry expertise. This work was supported by NCIR01CA151566, R01CA46413, 5I01BX001253, and P50CA095103 to RJC, T32CA119925 and American Gastroenterological Association Research Scholar Award to AEP, and T32GM008554 to E.J.P. Core Services performed through Vanderbilt University Medical Center\'s Digestive Disease Research Center supported by NIH Grant P30DK058404 Core Scholarship. The authors thank Ardeth and Ron Obenauf for their generous support.