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. 2014 Dec 3;16:487. doi: 10.1186/s13058-014-0487-6

Figure 2.

Figure 2

Embryonically EdU-labeled cells persist in the mammary ducts of adult mice. (A) Schematic protocol to study embryonic label retention in the mouse mammary gland. (B) EdU label incorporation was examined in paraffin sections from e15 embryos labeled for 24 hours. EdU+ cells are located in the epithelial bud and surrounding mesenchyme. Paraffin sections stained for EdU from MGs of newborn (C), three-week-old (D), and eight-week-old (E) mice after EdU administration between e14.5 and e18. L = lumen. (F) Percentage of EdU-labeled cells at various time-points after embryonic administration. (n = 3 to 5 mice/stage) (G) Diagram of LLRC location in eight week MG. (H) Percentage of EdU+ cells that express basal or luminal lineage markers in eight week MGs. (n = 1,000 cells). Bars represent SE. [See Additional file 2]. (I) Paraffin section of proximal portion of 10-week chased MG co-stained for EdU, K14 and Gata3. Edu + cells (red) are located predominantly in the basal layer of the ducts and are a subpopulation of the K14-positive cells (white; see yellow arrows). EdU+ cells are also located in the luminal layer of the ducts and are a rare subpopulation of the Gata3-positive cells (green; see white asterisks). (J-M) Summary of flow cytometry data. (J) MGs were isolated from eight-week-old EdU pulse-chased mice and analyzed by flow cytometry for expression of cell surface markers, CD24 and CD49f. EdU+ cells represent 1.2% of total mammary epithelial cells Lin-CD24+ [See Additional file 1]. (K) Immunofluorescence analysis for EdU+ cells in cytospins of sorted Lin-CD24+CD49flow luminal and Lin-CD24+CD49fhigh basal cells. (L) Representative histogram FACS plot of sorted and fixed populations of Lin-CD24+CD49flow luminal and Lin-CD24+CD49fhigh basal cells. (M) Average percentage of EdU+ cells in Lin-CD24+CD49flo luminal (2.2%) and Lin-CD24+CD49fhigh basal (8.88%) cell populations. Data represent mean ± SEM of three independent experiments. Scale bars, 20 μm, mm = mammary mesenchyme. e, embryonic day; EdU, 5-ethynl-2′-deoxyuridine; FACS, fluorescence activated cell sorting; LLRC, long label retaining cells; MG, mammary gland; SE, standard error; SEM, standard error of the mean.