rabbit monoclonal anti-progesterone receptor (pgr) (sp2, catalogue number: sab5500165-100ul, tested concentration at 1:400  (Millipore)

Journal: Frontiers in Endocrinology

Article Title: Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy

doi: 10.3389/fendo.2022.846226

Figure Lengend Snippet: List of Taqman probes used for RT-qPCR.

Article Snippet: Tissue cryosections (5µm) were prepared and stained using the following antibodies: rabbit monoclonal anti-progesterone receptor (PGR) (SP2, catalogue number: SAB5500165-100UL, tested concentration at 1:400, Sigma-Aldrich, St. Louis, MO); cytokeratin 8 (cTEC marker) (Troma-1, 1mL supernatant, stock concentration: 50ng/mL; tested concentration: 1ng/mL, Developmental Studies Hybridoma Bank, IA, USA), and cytokeratin 5 (mTEC marker) (stock concentration: 1mg/mL; tested concentration: 2ug/mL, Biolegend, San Diego, CA).

Techniques:

Journal: Frontiers in Endocrinology

Article Title: Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy

doi: 10.3389/fendo.2022.846226

Figure Lengend Snippet: Nuclear Progesterone Receptor expression increases in the thymus in pregnancy. (A) RT-qPCR for Esr1, Esr2, Pgr, Pgrmc1 , and Pgrmc2 between NP (n=3) and P (GD16.5, n=5) thymus. Each dot represents one mouse. (B) % PR positive nuclei calculated from field of DAPI from NP (n=4), GD6.5 (n=3), GD14.5 (n=6), GD16.5 (n=3), and GD18.5 (n=4) thymus; see Supplementary Figure 1 for representative images. *p < 0.05, ***p < 0.0001; one-way ANOVA with Dunnett’s post-hoc test with comparisons to NP controls. Each dot represents one mouse. (C) Immunofluorescence staining of GD14.5 thymus with antibodies specific to cytokeratin 8 (K8) and nuclear PGR. C, cortex; M, medulla, Scale bar = 100μm, 200x magnification.

Article Snippet: Tissue cryosections (5µm) were prepared and stained using the following antibodies: rabbit monoclonal anti-progesterone receptor (PGR) (SP2, catalogue number: SAB5500165-100UL, tested concentration at 1:400, Sigma-Aldrich, St. Louis, MO); cytokeratin 8 (cTEC marker) (Troma-1, 1mL supernatant, stock concentration: 50ng/mL; tested concentration: 1ng/mL, Developmental Studies Hybridoma Bank, IA, USA), and cytokeratin 5 (mTEC marker) (stock concentration: 1mg/mL; tested concentration: 2ug/mL, Biolegend, San Diego, CA).

Techniques: Expressing, Quantitative RT-PCR, Immunofluorescence, Staining

Journal: Frontiers in Endocrinology

Article Title: Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy

doi: 10.3389/fendo.2022.846226

Figure Lengend Snippet: Pgr reporter system identifies PGR + cells in the cortex of the thymus in pregnancy. (A) Explanation of Cre/Rosa26mTmG reporter system. In the absence of Cre recombinase activity, all cells remain mT+ (red). Upon expression of Pgr gene, Cre recombinase generation mediates the excision of the mT cassette. Post recombination, the cell membrane of all PGR+ cells will become GFP+. (B) Macroscopic visualization of GFP expression in the thymus at GD14.5 compared to virgin control. (C) Partially stitched Pgr cre/+ ;Rosa26 mTmG/mTmG thymus at GD14.5 to demonstrate GFP+ expression in the cortex of the thymus. K5 denotes medulla. DAPI = nuclei, PGR = GFP+ cells. The dotted lines outline the medulla. Magnification: 200x, Scale bar = 100μm. (D) Pgr cre/+ ;Rosa26 mTmG/mTmG thymus at GD14.5 stained with anti-PGR primary antibody and counter stained with anti-rabbit secondary antibody (Alexa Fluor 405), pseudo-colored red to provide contrast to GFP+ cells. Dotted line indicates the capsule of the thymus. Magnification: 200x, Scale bar = 100μm.

Article Snippet: Tissue cryosections (5µm) were prepared and stained using the following antibodies: rabbit monoclonal anti-progesterone receptor (PGR) (SP2, catalogue number: SAB5500165-100UL, tested concentration at 1:400, Sigma-Aldrich, St. Louis, MO); cytokeratin 8 (cTEC marker) (Troma-1, 1mL supernatant, stock concentration: 50ng/mL; tested concentration: 1ng/mL, Developmental Studies Hybridoma Bank, IA, USA), and cytokeratin 5 (mTEC marker) (stock concentration: 1mg/mL; tested concentration: 2ug/mL, Biolegend, San Diego, CA).

Techniques: Activity Assay, Expressing, Staining

Journal: Frontiers in Endocrinology

Article Title: Nuclear Progesterone Receptor Expressed by the Cortical Thymic Epithelial Cells Dictates Thymus Involution in Murine Pregnancy

doi: 10.3389/fendo.2022.846226

Figure Lengend Snippet: Conditional deletion of Pgr gene from the Foxn1+ thymic epithelial cells prevents thymic involution in pregnancy. (A) Nuclear progesterone receptor (PGR) colocalizes with Foxn1+ Thymic epithelial cells (TECs) at GD6.5. Alexa flour 405 was used as a secondary antibody. PGR is pseudo-colored red to provide contrast to the GFP+. 400x magnification, scale bar = 50μm. (B) Immunofluorescence staining to detect PGR protein confirms the conditional deletion of Pgr in the thymus at GD16.5 of Pgr d/d females. PGR expression is intact in the NP female uterus of both Pgr f/f controls and Pgr d/d animals. Magnification at 200x. Scale bar = 100μm, (C) Thymic weight (Top) and cellularity (Bottom) in NP and GD16.5 C57BL6WT, Pgr f/f / Pgr f/+ (FLFL/FLWT) or Pgr d/d (FLKO) females. Red dots = GD14.5. (D) Thymic weight (Top) and cellularity (Bottom) in C57BL6WT, Pgr f/f / Pgr f/+ (FLFL/FLWT) or Pgr d/d (FLKO) between NP and pregnant (GD14.5 - GD16.5) females. Each dot represents one mouse. Statistical analysis was performed using non-parametric Wilcoxon test (C) or Kruskal-Wallis test followed by Dunn’s test for post-hoc assessment with Bonferroni correction (D) . P<0.05 deemed significant. **p < 0.001, ***p < 0.0005, ****p < 0.00001, ns, non-significant.

Article Snippet: Tissue cryosections (5µm) were prepared and stained using the following antibodies: rabbit monoclonal anti-progesterone receptor (PGR) (SP2, catalogue number: SAB5500165-100UL, tested concentration at 1:400, Sigma-Aldrich, St. Louis, MO); cytokeratin 8 (cTEC marker) (Troma-1, 1mL supernatant, stock concentration: 50ng/mL; tested concentration: 1ng/mL, Developmental Studies Hybridoma Bank, IA, USA), and cytokeratin 5 (mTEC marker) (stock concentration: 1mg/mL; tested concentration: 2ug/mL, Biolegend, San Diego, CA).

Techniques: Immunofluorescence, Staining, Expressing

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Time-lapse imaging reveals prolonged roundup duration in Sp2−/− NPCs. (A) Schematic for generation and imaging of organotypic slice cultures from MADM cortices. Photomicrograph of an Emx1:MADM:wt/wt forebrain section at E14.5. (B) Schematic of mitotic NPCs with the clearly identifiable stages in our preparations marked by the blue bar. (C) Representative panels from live imaging experiments in control Emx1:MADM:wt/wt cortices. tdTomato-WT (red) and EGFP-WT (green) NPCs exhibited similar roundup durations. (D) Representative panels from Emx1:MADM:F/+ live imaging experiments. Green Sp2−/− progenitors spent more time in roundup than their sibling red Sp2+/+ cells. Elapsed time is indicated in minutes above each frame. Blue bars indicate frames in roundup. (E) Comparison of roundup durations indicates a significant increase in Sp2−/− (green) cells of Emx1:MADM:F/+ cortices compared with all other cell genotypes. Emx1:MADM:wt/wt: red, n=40 cells; green, n=45 cells. Emx1:MADM:F/+: red, n=56; green, n=59 cells; four independent litters analyzed for each genotype. One-way ANOVA, F=7.733; P<0.0001, *P<0.05 after Bonnferroni's multiple comparison correction. Scale bars: 500 µm in A; 10 µm in C,D. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Imaging

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs have prolonged early mitosis and decreased metaphase durations. (A) Schematic of in utero electroporation of an H2B::mCherry construct to visualize chromatin in en face time-lapse imaging of organotypic whole-mount preparations. (B) Representative images of cortical NPCs with time noted in minutes above each frame. Prophase+prometaphase (purple) and metaphase (orange) periods are marked as indicated by bars underneath the panels. (C) Quantification of time spent in each stage of mitosis. Sp2F/F (control), n=65 cells from three experiments; Emx1:F/F, n=60 cells from four experiments; prophase+prometaphase, t=2.403, d.f.=123 P=0.02. Metaphase: t=3.75, d.f.=123, P=0.0003. Data are mean±s.e.m. Scale bars: 10 µm. *P<0.05, Student's t-test.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: In Utero, Electroporation, Construct, Imaging

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Population analysis of control and Sp2−/− cells in the E14.5 MADM cortices. (A) Representative images of E14.5 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and nuclei counterstained with DAPI (blue). Scale bar: 50 µm. (B) Overall green to red ratios (G:R) for total number of cells, and by layer in the developing cortex. VZ/SVZ, ventricular/subventricular zone; IZ, intermediate zone; CP, cortical plate. Total cells, t=1.785, P=0.11; VZ/SVZ, t=3.119, P=0.01; IZ, t=1.196, P=0.27; CP, t=0.4838, P=0.64. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. (C) Average densities (values ×104/mm3) of MADM cells overall and by layer in the developing cortex. All layers: Emx1:MADM:wt/wt, t=1.883, P=0.10; Emx1:MADM:F/+, t=0.3231, P=0.75. VZ/SVZ: Emx1:MADM:wt/wt, t=6.866, P=0.0001; Emx1:MADM:F/+, t=1.383, P=0.20. IZ: Emx1:MADM:wt/wt, t=1.346, P=0.22; Emx1:MADM:F/+, t=0.1167, P=0.91. CP: Emx1:MADM:wt/wt, t=0.0754, P=0.94; Emx1:MADM:F/+, t=0.4735, P=0.65. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. Dots represent values from individual embryos. *P<0.05, Student's t-test. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs generate fewer subependymal and upper layer cells in the postnatal cortex. (A) Representative images of P0 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and co-stained for NeuN (blue) to delineate the cortical layers. Scale bar: 100 µm. (B) Green to red ratios (G:R) of the P0 cortex for all layers or by layer. SEZ, subpendymal zone; WM, white matter; DL, deep layers; UL, upper layers. All layers: t=6.458, P=0.0007; SEZ: t=3.143, P=0.02; WM: t=2.275, P=0.06; DL: t=1.41, P=0.21; UL: t=4.526, P=0.004; Student's t-test, *P<0.05; m=4 embryos per genotype. (C) Densities (values ×104/mm3) for MADM cells of the P0 cortex for all layers or by layer. All layers: Emx1:MADM:wt/wt, t=0.3883, P=0.71; Emx1:MADM:F/+, t=5.645, P=0.0013. SEZ: Emx1:MADM:wt/wt, t=0.7553, P=0.48; Emx1:MADM:F/+, t=5.753, P=0.0012. WM: Emx1:MADM:wt/wt, t=0.3416, P=0.74; Emx1:MADM:F/+, t=4.619, P=0.0036. DL: Emx1:MADM:wt/wt, t=0.3719, P=0.72; Emx1:MADM:F/+, t=0.8907, P=0.41. UL: Emx1:MADM:wt/wt, t=0.1627, P=0.88; Emx1:MADM:F/+, t=3.393, P=0.014. Student's t-test, *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Clonal analysis of cortical NPCs in the presence and absence of Sp2. (A) Schematic of experimental paradigm to induce individual cortical clones for analysis using the Nestin-creERT2 transgenic mouse that expresses tamoxifen-inducible cre under the control of the Nestin promoter (NcreER) in combination with MADM and floxed Sp2 alleles. Tamoxifen administration at E11.5 permits cre entry into the nucleus to induce highly sparse MADM recombination events in mitotic NPCs. Embryos were sacrificed at E18.5 for fixation, serial sectioning and immunohistochemistry to visualize and quantify clones. (B) Images of serial sections containing individual clones induced at E11.5 in NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos and harvested at E18.5. Scale bar: 50 µm. (C) Representative mapped clones reconstructed from NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos. (D) G minus R (G-R) values for total clone composition and by layer in the E18.5 cortex. All layers: NcreER:MADM:wt/wt, t=0.7405, P=0.46; NcreER:MADM:F/+, t=1.894, P=0.067. SEZ/WM: NcreER:MADM:wt/wt, t=0.4133, P=0.68; NcreER:MADM:F/+, t=1.738, P=0.091. DL: NcreER:MADM:wt/wt, t=0.415, P=0.68; NcreER:MADM:F/+, t=0.6746, P=0.50. UL: NcreER:MADM:wt/wt, t=0.8134, P=0.42; NcreER:MADM:F/+, t=2.865, P=0.0071. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. One-sample t-test. *P<0.05. (E) Laminar distribution of red and green MADM cells in individual clones (dots) by genotype. SEZ/WM: NcreER:MADM:wt/wt, t=0.9489, P=0.35; NcreER:MADM:F/+, t=0.6531, P=0.43. DL: NcreER:MADM:wt/wt, t=0.1238, P=0.9; NcreER:MADM:F/+, t=0.25, P=0.80. UL: NcreER:MADM:wt/wt, t=1.157, P=0.251; NcreER:MADM:F/+, t=1.851, P=0.067. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. Student's t-test. *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Clone Assay, Transgenic Assay, Immunohistochemistry

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2 is dispensable for proliferation and expansion of NSCs. (A) Representative time-lapse en face images of FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortical wholemounts (time indicated in minutes above each panel). Arrows indicate cells tracked for each genotype and blue bars indicate frames in roundup. Scale bar: 10 µm. (B) Quantification of the time spent in roundup. FoxG1:MADM:wt/wt: red, n=30 cells; green, n=30 cells. FoxG1:MADM:F/+: red, n=34 cells; green, n=37 cells. One-way ANOVA, F=1.828, P=0.15. (C) Representative images of E10.5 FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortices immunoenhanced for EGFP (green) and tdTomato (red). Nuclei are counterstained with DAPI (blue). Scale bar: 25 µm. (D) G:R for cells in E10.5 cortices, t=1.113, P=0.33, n=3 embryos per genotype. (E) Red and green cell density; density values are ×104/mm3. FoxG1:MADM:wt/wt, t=2.082, P=0.11; FoxG1:MADM:F/+, t=1.519, P=0.20. n=3 animals per genotype from three independent litters. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NSCs proliferate and differentiate in the E10.5 cortex. (A) Sample western blot of E10.5 cortical lysates blotted for Sp2 and actin shows depletion of Sp2 in FoxG1:F/+ and FoxG1:F/F cortices compared with Sp2F/F controls. Weight markers are in kilodaltons (kDa). (B) E10.5 cortices counterstained with DAPI for comparison of average width in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=6) mice; t=0.5565, P=0.59. Scale bar: 100 µm. (C) High-power confocal images of E10.5 cortices immunostained for PH3 and counterstained with DAPI. Quantification of PH3 densities in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=5) cortices; t=1.147, P=0.28. (D) Spindle angle distribution of PH3+ cells in late anaphase or early telophase, as indicated by bins (percent per segment). Angles were measured for the cleavage planes relative to the apical surface. FoxG1:wt/wt, n=33; FoxG1:F/F, n=42; t=0.9107, P=0.37. (E) High-power images of E10.5 cortices immunostained for Tbr2 and counterstained with DAPI. Quantification of Tbr2 densities in FoxG1:wt/wt (n=3) and FoxG1:F/F (n=3) cortices; t=0.8435, P=0.45. (F) High-power images of E10.5 cortices immunostained for Tuj1 and counterstained with DAPI. Quantification of Tuj1 densities in FoxG1:wt/wt (n=4) and FoxG1:F/F (n=5) cortices; t=0.8903, P=0.40. Densities are values ×105 cells/mm3. Dots in charts represent values for individual embryos. Scale bars: 25 µm in C,E,F. Embryos were from at least three independent litters for each genotype. Student's t-test. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Antibodies Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Western Blot

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Time-lapse imaging reveals prolonged roundup duration in Sp2−/− NPCs. (A) Schematic for generation and imaging of organotypic slice cultures from MADM cortices. Photomicrograph of an Emx1:MADM:wt/wt forebrain section at E14.5. (B) Schematic of mitotic NPCs with the clearly identifiable stages in our preparations marked by the blue bar. (C) Representative panels from live imaging experiments in control Emx1:MADM:wt/wt cortices. tdTomato-WT (red) and EGFP-WT (green) NPCs exhibited similar roundup durations. (D) Representative panels from Emx1:MADM:F/+ live imaging experiments. Green Sp2−/− progenitors spent more time in roundup than their sibling red Sp2+/+ cells. Elapsed time is indicated in minutes above each frame. Blue bars indicate frames in roundup. (E) Comparison of roundup durations indicates a significant increase in Sp2−/− (green) cells of Emx1:MADM:F/+ cortices compared with all other cell genotypes. Emx1:MADM:wt/wt: red, n=40 cells; green, n=45 cells. Emx1:MADM:F/+: red, n=56; green, n=59 cells; four independent litters analyzed for each genotype. One-way ANOVA, F=7.733; P<0.0001, *P<0.05 after Bonnferroni's multiple comparison correction. Scale bars: 500 µm in A; 10 µm in C,D. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Imaging, Control, Comparison

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs have prolonged early mitosis and decreased metaphase durations. (A) Schematic of in utero electroporation of an H2B::mCherry construct to visualize chromatin in en face time-lapse imaging of organotypic whole-mount preparations. (B) Representative images of cortical NPCs with time noted in minutes above each frame. Prophase+prometaphase (purple) and metaphase (orange) periods are marked as indicated by bars underneath the panels. (C) Quantification of time spent in each stage of mitosis. Sp2F/F (control), n=65 cells from three experiments; Emx1:F/F, n=60 cells from four experiments; prophase+prometaphase, t=2.403, d.f.=123 P=0.02. Metaphase: t=3.75, d.f.=123, P=0.0003. Data are mean±s.e.m. Scale bars: 10 µm. *P<0.05, Student's t-test.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: In Utero, Electroporation, Construct, Imaging, Control

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Population analysis of control and Sp2−/− cells in the E14.5 MADM cortices. (A) Representative images of E14.5 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and nuclei counterstained with DAPI (blue). Scale bar: 50 µm. (B) Overall green to red ratios (G:R) for total number of cells, and by layer in the developing cortex. VZ/SVZ, ventricular/subventricular zone; IZ, intermediate zone; CP, cortical plate. Total cells, t=1.785, P=0.11; VZ/SVZ, t=3.119, P=0.01; IZ, t=1.196, P=0.27; CP, t=0.4838, P=0.64. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. (C) Average densities (values ×104/mm3) of MADM cells overall and by layer in the developing cortex. All layers: Emx1:MADM:wt/wt, t=1.883, P=0.10; Emx1:MADM:F/+, t=0.3231, P=0.75. VZ/SVZ: Emx1:MADM:wt/wt, t=6.866, P=0.0001; Emx1:MADM:F/+, t=1.383, P=0.20. IZ: Emx1:MADM:wt/wt, t=1.346, P=0.22; Emx1:MADM:F/+, t=0.1167, P=0.91. CP: Emx1:MADM:wt/wt, t=0.0754, P=0.94; Emx1:MADM:F/+, t=0.4735, P=0.65. Emx1:MADM:wt/wt, n=5; Emx1:MADM:F/+, n=5. Dots represent values from individual embryos. *P<0.05, Student's t-test. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Control

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NPCs generate fewer subependymal and upper layer cells in the postnatal cortex. (A) Representative images of P0 Emx1:MADM:wt/wt and Emx1:MADM:F/+ cortices immunoenhanced for EGFP (green), tdTomato (red) and co-stained for NeuN (blue) to delineate the cortical layers. Scale bar: 100 µm. (B) Green to red ratios (G:R) of the P0 cortex for all layers or by layer. SEZ, subpendymal zone; WM, white matter; DL, deep layers; UL, upper layers. All layers: t=6.458, P=0.0007; SEZ: t=3.143, P=0.02; WM: t=2.275, P=0.06; DL: t=1.41, P=0.21; UL: t=4.526, P=0.004; Student's t-test, *P<0.05; m=4 embryos per genotype. (C) Densities (values ×104/mm3) for MADM cells of the P0 cortex for all layers or by layer. All layers: Emx1:MADM:wt/wt, t=0.3883, P=0.71; Emx1:MADM:F/+, t=5.645, P=0.0013. SEZ: Emx1:MADM:wt/wt, t=0.7553, P=0.48; Emx1:MADM:F/+, t=5.753, P=0.0012. WM: Emx1:MADM:wt/wt, t=0.3416, P=0.74; Emx1:MADM:F/+, t=4.619, P=0.0036. DL: Emx1:MADM:wt/wt, t=0.3719, P=0.72; Emx1:MADM:F/+, t=0.8907, P=0.41. UL: Emx1:MADM:wt/wt, t=0.1627, P=0.88; Emx1:MADM:F/+, t=3.393, P=0.014. Student's t-test, *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Staining

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Clonal analysis of cortical NPCs in the presence and absence of Sp2. (A) Schematic of experimental paradigm to induce individual cortical clones for analysis using the Nestin-creERT2 transgenic mouse that expresses tamoxifen-inducible cre under the control of the Nestin promoter (NcreER) in combination with MADM and floxed Sp2 alleles. Tamoxifen administration at E11.5 permits cre entry into the nucleus to induce highly sparse MADM recombination events in mitotic NPCs. Embryos were sacrificed at E18.5 for fixation, serial sectioning and immunohistochemistry to visualize and quantify clones. (B) Images of serial sections containing individual clones induced at E11.5 in NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos and harvested at E18.5. Scale bar: 50 µm. (C) Representative mapped clones reconstructed from NcreER:MADM:wt/wt and NcreER:MADM:F/+ embryos. (D) G minus R (G-R) values for total clone composition and by layer in the E18.5 cortex. All layers: NcreER:MADM:wt/wt, t=0.7405, P=0.46; NcreER:MADM:F/+, t=1.894, P=0.067. SEZ/WM: NcreER:MADM:wt/wt, t=0.4133, P=0.68; NcreER:MADM:F/+, t=1.738, P=0.091. DL: NcreER:MADM:wt/wt, t=0.415, P=0.68; NcreER:MADM:F/+, t=0.6746, P=0.50. UL: NcreER:MADM:wt/wt, t=0.8134, P=0.42; NcreER:MADM:F/+, t=2.865, P=0.0071. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. One-sample t-test. *P<0.05. (E) Laminar distribution of red and green MADM cells in individual clones (dots) by genotype. SEZ/WM: NcreER:MADM:wt/wt, t=0.9489, P=0.35; NcreER:MADM:F/+, t=0.6531, P=0.43. DL: NcreER:MADM:wt/wt, t=0.1238, P=0.9; NcreER:MADM:F/+, t=0.25, P=0.80. UL: NcreER:MADM:wt/wt, t=1.157, P=0.251; NcreER:MADM:F/+, t=1.851, P=0.067. NcreER:MADM:wt/wt, n=37; NcreER:MADM:F/+, n=35 clones. Student's t-test. *P<0.05. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Clone Assay, Transgenic Assay, Control, Immunohistochemistry

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2 is dispensable for proliferation and expansion of NSCs. (A) Representative time-lapse en face images of FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortical wholemounts (time indicated in minutes above each panel). Arrows indicate cells tracked for each genotype and blue bars indicate frames in roundup. Scale bar: 10 µm. (B) Quantification of the time spent in roundup. FoxG1:MADM:wt/wt: red, n=30 cells; green, n=30 cells. FoxG1:MADM:F/+: red, n=34 cells; green, n=37 cells. One-way ANOVA, F=1.828, P=0.15. (C) Representative images of E10.5 FoxG1:MADM:wt/wt and FoxG1:MADM:F/+ cortices immunoenhanced for EGFP (green) and tdTomato (red). Nuclei are counterstained with DAPI (blue). Scale bar: 25 µm. (D) G:R for cells in E10.5 cortices, t=1.113, P=0.33, n=3 embryos per genotype. (E) Red and green cell density; density values are ×104/mm3. FoxG1:MADM:wt/wt, t=2.082, P=0.11; FoxG1:MADM:F/+, t=1.519, P=0.20. n=3 animals per genotype from three independent litters. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques:

Journal: Development (Cambridge, England)

Article Title: Sp2 regulates late neurogenic but not early expansive divisions of neural stem cells underlying population growth in the mouse cortex

doi: 10.1242/dev.186056

Figure Lengend Snippet: Sp2−/− NSCs proliferate and differentiate in the E10.5 cortex. (A) Sample western blot of E10.5 cortical lysates blotted for Sp2 and actin shows depletion of Sp2 in FoxG1:F/+ and FoxG1:F/F cortices compared with Sp2F/F controls. Weight markers are in kilodaltons (kDa). (B) E10.5 cortices counterstained with DAPI for comparison of average width in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=6) mice; t=0.5565, P=0.59. Scale bar: 100 µm. (C) High-power confocal images of E10.5 cortices immunostained for PH3 and counterstained with DAPI. Quantification of PH3 densities in FoxG1:wt/wt (n=5) and FoxG1:F/F (n=5) cortices; t=1.147, P=0.28. (D) Spindle angle distribution of PH3+ cells in late anaphase or early telophase, as indicated by bins (percent per segment). Angles were measured for the cleavage planes relative to the apical surface. FoxG1:wt/wt, n=33; FoxG1:F/F, n=42; t=0.9107, P=0.37. (E) High-power images of E10.5 cortices immunostained for Tbr2 and counterstained with DAPI. Quantification of Tbr2 densities in FoxG1:wt/wt (n=3) and FoxG1:F/F (n=3) cortices; t=0.8435, P=0.45. (F) High-power images of E10.5 cortices immunostained for Tuj1 and counterstained with DAPI. Quantification of Tuj1 densities in FoxG1:wt/wt (n=4) and FoxG1:F/F (n=5) cortices; t=0.8903, P=0.40. Densities are values ×105 cells/mm3. Dots in charts represent values for individual embryos. Scale bars: 25 µm in C,E,F. Embryos were from at least three independent litters for each genotype. Student's t-test. Dots represent values from individual embryos. Data are mean±s.e.m.

Article Snippet: Primary antibodies used were: anti-Actin-HRP (Santa Cruz Biotechnology, sc-1615, 1:5000), anti-GAPDH-HRP (Santa Cruz Biotechnology, sc20357, 1:5000), chicken anti-GFP (Abcam, ab13970, 1:2000), rabbit anti-RFP (Abcam, ab62341, 1:500), mCherry (Abcam, ab167453, 1:1000), rabbit anti-Tbr2 (Abcam, ab23345, 1:300), mouse anti-Tuj1 (Covance, 801201, 1:500), mouse anti-Pax6 (Chemicon, MAB5554, 1:500), rabbit anti-Pax6 (Covance, 901301, 1:500), mouse anti-PH3 (Abcam, ab14955, 1:1000), rabbit anti-Sp2 (Sigma-Aldrich, HPA003357, 1:1000), rabbit anti-Sp2 (Novus, NB100-92358, 1:1000) and mouse anti-NeuN (Millipore, MAB377, 1:1000).

Techniques: Western Blot, Comparison

Journal: Human Reproduction (Oxford, England)

Article Title: Endometrial stromal cell attachment and matrix homeostasis in abdominal wall endometriomas

doi: 10.1093/humrep/dex371

Figure Lengend Snippet: Antibodies for immunohistochemistry.

Article Snippet: Negative controls were comprised of all treatments but without primary antibody. table ft1 table-wrap mode="anchored" t5 Table I caption a7 Antigen Clone Dilution Source Species Estrogen receptor α SP1 1:25 Spring Bioscience, Pleasanton, CA Rabbit Pax8 NA-Polyclonal 1:1600 Proteintech, Rosemont, IL Rabbit Progesterone receptor SP2 1:400 LifeSpan Biosciences Rabbit mAb Smad3 C67H9 1:1000 Cell Signaling Rabbit mAb Phosphor-Smad3 C25A9 1:1000 Cell Signaling Rabbit mAb CD68 PGM1 1:100 Invitrogen Mouse mAb Integrin α5 NA 1:250 Abcam Rabbit mAb Integrin β1 NA 1:500 Abcam Rabbit mAb Open in a separate window Antibodies for immunohistochemistry.

Techniques: Immunohistochemistry