mouse anti-p-rb  (Santa Cruz Biotechnology)

Journal: eLife

Article Title: Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland

doi: 10.7554/eLife.93326

Figure Lengend Snippet: ( A ) Representative 3D surface rendering images of EpCAM-stained mammary glands of Krt14-Eda embryos and their wild type (WT) littermates at E13.5 and E14.5. Mammary gland 2 is shown. Ectopic mammary rudiments (asterisk) common in Krt14-Eda embryos were excluded from the analysis. Scale bar, 20 µm. ( B, C ) Quantification of mammary gland volume ( B ) and branching tip number ( C ) at E13.5 (n WT = 17, n Krt14-Eda =21) and at E14.5 (n WT = 27 and n Krt14-Eda =22). ( D, E ) Quantification of the proportions of mammary epithelial cells in S/G2/M and G1/G0 phases in the entire epithelium ( D ) and the proportions of mammary epithelial cells in S/G2/M phase in basal and inner compartments ( E ) in WT or Krt14-Eda embryos at E13.5 (n WT = 17 glands and in total 7714 cells from three embryos, n Krt14-Eda =21 glands and in total 15,561 cells from 4 embryos) and E14.5 (n WT = 16 glands and in total 10,221 cells from 4 embryos, n Krt14-Eda =18 glands and in total 10,520 cells from 5 embryos). ( F ) Representative images showing the growth of E13.5 and E14.5 Krt14-Eda and wild type littermate epithelial mammary rudiments in 3D Matrigel culture. Note branching in E14.5 Krt14-Eda mammary rudiments. Scale bar, 500 µm. ( G ) Quantification of branching mammary rudiments in 3D culture. Data are presented as percentage of branching mammary rudiments (mean ± SD) from a total of 5 (E13.5 WT), 6 (E13.5 Krt14-Eda), 3 (E14.5 WT) and 3 (E14.5 Krt14-Eda) independent experiments (each with minimum 5 rudiments in culture). ( H ) Representative 3D surface rendering images of EpCAM-stained E15.5 and E16.5 epithelial mammary rudiments of Eda -/- and wild type embryos. Mammary gland 2 is shown. Scale bar, 50 µm. ( I, J ) Quantification of epithelial mammary gland volume ( I ) and number of branching tips ( J ), at E15.5 (n WT = 17 and n Eda-/- = 33) and at E16.5 (n WT = 32 and n Eda-/- = 68). ( K, L ) Quantification of the proportions of mammary epithelial cells in S/G2/M or G1/G0 phases ( K ) and the proportions of mammary epithelial cells in S/G2/M phase in basal and inner compartments ( L ) in WT or Eda -/- embryos at E15.5 (n WT = 17 glands and in total 14,054 cells from 3 embryos, n Eda-/- = 27 glands and in total 21,986 cells from 5 embryos) and E16.5 (n WT = 34 glands and in total 72,279 cells from 3 embryos, n Eda-/- = 64 glands and in total 76,844 cells from 3 embryos). ( M ) Representative images showing E15.5 and E16.5 Eda -/- and wild type epithelial mammary rudiments in 3D culture after 3 days. Scale bar, 200 µm. ( N ) Quantification of branching mammary rudiments in 3D culture. Data are presented as percentage of branching mammary rudiments from a total of 10 WT and 19 Eda -/- E16.5 embryos (each with 3–6 rudiments in culture). Data are presented as mean ± SD. The statistical significance was assessed using unpaired two-tailed Student’s t -test with Bonferroni correction, except Wilcoxon test with Bonferroni correction for ( C , G and J ). ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 3—source data 1. Source data of quantifications represented as graphs in .

Article Snippet: The following antibodies were used in this study: rat anti-mouse CD326 (EpCAM, 552370, BD Pharmingen, 1:500), rabbit anti-mouse Krt14 (RB-9020-P, Thermo Fisher Scientific, 1:500), rabbit anti-cleaved Caspase-3 (9661, Cell Signaling Technology, 1:500), Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, 1:500) and Alexa Fluor 647-conjugated Donkey anti-Rat secondary antibody (A48272, Invitrogen, 1:500).

Techniques: Staining, Two Tailed Test

Journal: eLife

Article Title: Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland

doi: 10.7554/eLife.93326

Figure Lengend Snippet: ( A ) Confocal optical sections of whole-mount mammary glands from E13.5 and E14.5 Krt14-Eda or WT littermate embryos expressing Fucci2a reporter stained with EpCAM. Scale bars, 20 µm (E13.5) and 50 µm (E14.5). ( B ) Confocal optical sections of whole-mount mammary glands from E15.5 and E16.5 WT or Eda -/- Fucci2a embryos stained with EpCAM. Scale bars, 20 µm (E15.5) or 50 µm (E16.5). ( C ) Representative 3D surface rendering images and bud volume quantification of EpCAM-stained mammary gland 2 from WT or Eda -/- embryos at E13.5 (n WT = 8, n Eda-/- = 13). Scale bars, 20 µm. ( D, E ) Representative optical sections showing the 3D segmentation and cellular volume quantification of epithelial cells of mammary gland 2 from WT or Eda -/- embryos at E13.5 (n WT = 5 glands with total 3385 cells, n Eda-/- = 5 glands with total 2369 cells) ( D ) and E15.5 (n WT = 6 glands with total 11,151 cells, n Eda-/- = 6 glands with total 7946 cells). ( E ). Scale bars, 20 µm. Data are presented as mean ± SD and the statistical significances were assessed using unpaired two-tailed Student’s t -test. *, p<0.05; **, p<0.01; ***, p<0.001. Figure 3—figure supplement 1—source data 1. Source data of quantifications represented as graphs in .

Article Snippet: The following antibodies were used in this study: rat anti-mouse CD326 (EpCAM, 552370, BD Pharmingen, 1:500), rabbit anti-mouse Krt14 (RB-9020-P, Thermo Fisher Scientific, 1:500), rabbit anti-cleaved Caspase-3 (9661, Cell Signaling Technology, 1:500), Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, 1:500) and Alexa Fluor 647-conjugated Donkey anti-Rat secondary antibody (A48272, Invitrogen, 1:500).

Techniques: Expressing, Staining, Two Tailed Test

Journal: eLife

Article Title: Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland

doi: 10.7554/eLife.93326

Figure Lengend Snippet: ( A ) Representative images of E16.5 Krt14-Cre;Rosa26 mTmG/+ mammary glands cultured ex vivo for three days in the presence of 150 ng/ml recombinant IGF-1 or vehicle (BSA). Scale bar, 200 µm. ( B ) Quantification of the number of branching tips in vehicle (n=33) and IGF-1 treated (n=40) mammary gland explants. Data are pooled from five independent experiments and presented as mean ± SD. ( C ) Body weight of Igf1r +/+ , Igf1r +/- and Igf1r -/- embryos at E16.5 (n Igf1r+/+ =10, n Igf1r+/- =16, n Igf1r-/- =7), and E18.5 (n Igf1r+/+ =20, n Igf1r+/- =20, n Igf1r-/- =17). ( D ) Representative images of EpCAM-stained ventral skin including mammary glands (MG) 1–5 from Igf1r +/+ , Igf1r +/- and Igf1r -/- female embryos at E16.5, and E18.5. Note absence of MG3 in Igf1r -/- embryos. Magnifications show mammary gland 2. Scale bars, 500 µm. ( E, F ) Quantification of mammary gland area ( E ) and number of branch tips ( F ) normalized to body weight in Igf1r +/+ , Igf1r +/- and Igf1r -/- embryos at E18.5. MG5 was often lost during dissection and therefore was not included in the analysis. n.d, not detected. ( G ) Representative 3D surface rendering images of EpCAM-stained mammary gland 2 from Igf1r +/+ and Igf1r -/- embryos at E13.5 (n Igf1r+/+ =7, n Igf1r-/- =6), E16.5 (n Igf1r+/+ =6, n Igf1r-/- =7), and E18.5 (n Igf1r+/+ =9, n Igf1r-/- =11), based on 3D confocal imaging. Scale bar, 100 µm. ( H–I ), Quantification of epithelial mammary gland volume ( H ) and volume normalized with body weight ( I ). Data are presented as mean ± SD. ( J, K ) Representative images ( J ) showing the growth of E16.5 mammary epithelia isolated from control ( Igf1r +/+ or Igf1r +/- ) or Igf1r -/- embryos recombined with E16.5 mammary mesenchyme from control or Igf1r -/- embryos, as indicated in each figure. Explants were cultured for 4 days and the epithelium visualized with EpCAM staining. Quantifications are shown in ( K ). Scale bar, 100 µm. Data are pooled from 6 independent experiments and presented as mean ± SD. n is indicated in the right corner of each image in ( J ). Statistical significances were assessed using unpaired two-tailed Student’s t -test for ( A ) or unpaired two-tailed Student’s t -test with Bonferroni correction for ( C, E, F, H, I and K ). ns, non-significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 7—source data 1. Source data of quantifications represented as graphs in .

Article Snippet: The following antibodies were used in this study: rat anti-mouse CD326 (EpCAM, 552370, BD Pharmingen, 1:500), rabbit anti-mouse Krt14 (RB-9020-P, Thermo Fisher Scientific, 1:500), rabbit anti-cleaved Caspase-3 (9661, Cell Signaling Technology, 1:500), Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, 1:500) and Alexa Fluor 647-conjugated Donkey anti-Rat secondary antibody (A48272, Invitrogen, 1:500).

Techniques: Cell Culture, Ex Vivo, Recombinant, Staining, Dissection, Imaging, Isolation, Control, Two Tailed Test

Journal: eLife

Article Title: Mesenchyme instructs growth while epithelium directs branching in the mouse mammary gland

doi: 10.7554/eLife.93326

Figure Lengend Snippet:

Article Snippet: The following antibodies were used in this study: rat anti-mouse CD326 (EpCAM, 552370, BD Pharmingen, 1:500), rabbit anti-mouse Krt14 (RB-9020-P, Thermo Fisher Scientific, 1:500), rabbit anti-cleaved Caspase-3 (9661, Cell Signaling Technology, 1:500), Alexa Fluor 488-conjugated Donkey anti-Rat secondary antibody (A21208, Invitrogen, 1:500) and Alexa Fluor 647-conjugated Donkey anti-Rat secondary antibody (A48272, Invitrogen, 1:500).

Techniques: Recombinant, Virus, Software