Triple immunostaining for GFP, p75, and PRRX1.

<p>Triple immunostaining for GFP (<i>green</i>), the neural crest marker p75 (<i>white</i>), and PRRX1 (<i>red</i>) was performed on E20.5. The boxed area in <b>A</b> is enlarged in <b>B–Bʹʹʹ</b>. GFP/p75/PRRX1-triple (<i>yellow arrowheads</i>), GFP/p75-double (<i>yellow open arrowheads</i>), and p75/PRRX1-double (<i>white open arrowheads</i>) positive cells are indicated. Counts of each cell type (n = 2, with two slices each) is listed in <b>C</b>. <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>A</b>) and10 μm (<b>B–Bʹʹʹ</b>).</p

Immunostaining for GFP, DESMIN, α-SMA and PRRX1 and staining with isolectin B4.

<p>Using sagittal sections of embryonic pituitaries on E18.5, staining for vascular endothelial cells with isolectin B4 (<b>Aʹ</b>) or α-SMA (<b>Dʹ</b>) was performed by visualization with Cy5 (<i>white</i>), together with staining of GFP (FITC; <i>green</i>, <b>A</b> and <b>B</b>) and the pericyte marker DESMIN or PRRX1 (Cy3; <i>red</i>, <b>Aʹʹ</b> and <b>Bʹʹ</b>). GFP/DESMIN-double (<i>yellow open arrowheads</i>), DESMIN/isolectin B4-double (<i>white open arrowheads</i>), GFP-single (<i>yellow arrows</i>), and DESMIN- or α-SMA-single (<i>white arrowheads</i>) positive cells are indicated. Merged images (<b>Aʹʹʹ</b>, <b>Bʹʹʹ</b>, <b>Dʹʹʹ</b>, <b>and Eʹʹʹ</b>) are shown on the right. Each cell type (n = 2 with two slices each) was counted, and results are listed in <b>C</b> and <b>E</b>. <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>Aʹʹʹ</b> and <b>Dʹʹʹ</b>) and 10 μm (<b>Bʹʹʹ</b> and <b>Eʹʹʹ</b>).</p

GFP-positive cells during pituitary development.

<p>Using sagittal sections of embryonic pituitaries on E18.5 (<b>A</b>–<b>Bʹʹʹ</b>) and E19.5 (<b>C</b>–<b>Eʹʹʹ</b>), immunostaining was performed for GFP, PRRX1, and SOX2, which were visualized with FITC (<i>green</i>), Cy3 (<i>red</i>), and Cy5 (<i>white</i>), respectively. GFP/PRRX1/SOX2-triple (<i>yellow arrowheads</i>), GFP/PRRX1-double (<i>yellow open arrowheads</i>), PRRX1/SOX2-double (<i>white arrowheads</i>), GFP-single (<i>yellow arrows</i>) and PRRX1-single (<i>white open arrowheads</i>) positive cells are indicated. The boxed areas in <b>A</b> and <b>C</b> are enlarged in <b>B</b>–<b>Bʹʹʹ</b>, <b>D</b>–<b>Dʹʹʹ</b>, and <b>E</b>–<b>Eʹʹʹ</b>. Each cell type was counted (n = 1 for E18.5, n = 2 for E19.5, and n = 2 for E20.5, with four slices each), and results are shown in <b>F</b>. <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>A</b>, <b>C</b>) and 10 μm (<b>B–Bʹʹʹ</b> and <b>D–Eʹʹʹ</b>).</p

Double immunostaining for GFP and Ki67.

<p>Double immunostaining for GFP (<i>green</i>) and Ki67 (<i>red</i>) was performed on a section on E20.5. The boxed area in <b>A</b> is enlarged in <b>B</b>–<b>Bʹʹ</b>. GFP/Ki67-double positive (<i>yellow open arrowheads</i>) and GFP-single positive (<i>yellow arrow</i>) cells are indicated. Each cell type was counted in the anterior lobe (n = 2, with one slice each), and results are shown in <b>C.</b> <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>A</b>) and 10 μm (<b>B–Bʹʹ</b>).</p

GFP-positive cells at Atwell's recess on E15.5.

<p>Using sagittal sections of embryonic pituitaries on E15.5, immunostaining was performed for GFP, PRRX1, and SOX2, which were visualized with FITC (<b>A</b> and <b>B</b>, <i>green</i>), Cy3 (<b>Aʹ</b> and <b>Bʹ</b>, <i>red</i>), and Cy5 (<b>Aʹʹ</b> and <b>Bʹʹ</b>, <i>white</i>), respectively. Cells positive for GFP only are indicated with yellow arrows. The boxed area in <b>Aʹʹʹ</b> is enlarged in <b>B</b>–<b>Bʹʹʹ</b>. Each cell type was counted in four independent pituitaries (n = 4), and results are listed in <b>C</b>. <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe; <i>AR</i> Atwell's recess. Bars = 50 μm (<b>A–Aʹʹʹ</b>) and 10 μm (<b>B–Bʹʹʹ</b>).</p

Triple immunostaining for GFP, pituitary hormones, and SOX2.

<p>Triple immunostaining using a section from E20.5 was performed for GFP (<i>green</i>), SOX2 (<i>red</i>), and pituitary hormones (<i>white</i>) using a cocktail of antibodies against the hormones FSHβ, LHβ, prolactin, TSHβ, ACTH, and GH (HORMONES). The boxed area in <b>A</b> is enlarged in <b>B</b>–<b>Bʹʹʹ</b> and <b>C</b>–<b>Cʹʹʹ</b>. GFP/SOX2-double positive (<i>yellow open arrowheads</i>), SOX2/HORMONES-double positive (<i>white arrowheads</i>), GFP-single positive (<i>yellow arrows</i>), and HORMONES-single positive (<i>white open arrowheads</i>) cells are indicated. <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>A</b>) and 10 μm (<b>B</b>–<b>Cʹʹʹ</b>).</p

The frequency of GFP/isolectin B4-double positive cells on E15.5 and 18.5.

<p>The frequency of GFP/isolectin B4-double positive cells on E15.5 and 18.5.</p

Double immunostaining of GFP and isolectin B4.

<p>Using sagittal sections of embryonic pituitaries on E15.5 (<b>A–Bʹʹ</b>), E18.5 (<b>C–Dʹʹ</b>), and E20.5 (<b>E–Hʹʹ</b>), immunostaining was performed on four slices for GFP and endothelial cell marker isolectin B4 and was visualized with FITC (<i>green</i>) and Cy5 (<i>white</i>), respectively. Merged images are shown on the right. The boxed areas in <b>Aʹʹ</b>, <b>Cʹʹ</b>, and <b>Eʹʹ</b> are enlarged in <b>B</b>–<b>Bʹʹ</b>, <b>D</b>–<b>Dʹʹ</b>, and <b>F</b>–<b>Fʹʹ</b>. GFP/isolectin B4-double (<i>yellow arrowheads</i>), GFP-single (<i>yellow arrows</i>), and isolectin B4-single (<i>open white arrowheads</i>) cells are indicated. The boxed area in <b>Fʹʹ</b> is enlarged in <b>G–Gʹʹ</b>, and its orthogonal projections were analyzed by confocal Z-stack imaging with 0.1-μm slices (<b>H</b>–<b>Hʹʹ</b>). <i>AL</i> anterior lobe; <i>IL</i> intermediate lobe; <i>PL</i> posterior lobe. Bars = 50 μm (<b>Aʹʹ</b>, <b>Cʹʹ</b>, <b>Eʹʹ</b>, and <b>Gʹʹ</b>) and 10 μm (<b>Bʹʹ</b>, <b>Dʹʹ</b>, <b>Fʹʹ</b>, and <b>Hʹʹ</b>).</p