Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: The spatiotemporal relationship of PDGFR-β and two intermediate filament proteins nestin and vimentin in the lesioned striatum at days 3 and 7 after 3-NP injection. (A,B,E,F) Triple-labeling for PDGFR-β, GFAP and nestin at day 3 post-lesion showing that vascular profiles expressing PDGFR-β are positive for nestin in the lesion core (right side of the broken line), which is devoid of GFAP immunoreactivity. Note that nestin-positive cells in the peri-lesional area are astrocytes. (C,D,G,H) At 7 days, distribution of PDGFR-β in the vascular profiles overlaps well with that of nestin in the lesion core (right side of the broken line). (E–H) Higher magnification images of the boxed areas in (A–D) , respectively. (I–K) Triple-labeling for nestin, Ki-67 and GFAP. Note that a pair of nestin-positive cells that appear to be daughter cells are positive for Ki-67. (L) 5-Bromo-2-deoxyuridine (BrdU) injection protocol. (M,N) Triple-labeling for nestin, BrdU and GFAP showing that PDGFR-β/nestin double-positive cells within vascular profiles exhibit BrdU-labeled cell nuclei. The boxed area in (M) is enlarged in (N) . (O–R) Triple labeling for PDGFR-β, nestin and vimentin showing that nearly all of PDGFR-β/nestin double-positive cells co-express vimentin. Cell nuclei are stained with DAPI. Scale bars = 100 μm for (A–D) ; 20 μm for (E–H,I,J–M) ; 10 μm for (N) ; 50 μm for (O–R) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Injection, Labeling, Expressing, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Characterization of extravascular PDGFR-β-positive cells in the lesioned striatum at days 14 and 28 after 3-NP injection. (A–H) Triple labeling for PDGFR-β, GFAP and nestin at day 14 (A,C–E) and 28 (B,F–H) post-lesion showing that the distribution of PDGFR-β overlaps well with that of nestin. Note that PDGFR-β-positive processes show finer intertwined profiles, while nestin staining reveals distinct fibrous structures. (C–H) Higher magnification images of the boxed areas in (A,B) , respectively. (I–N) Double labeling for nestin and either laminin (I–K) or fibronectin (L–N) , showing that nestin is colocalized with two stromal cell markers. (O,Q–T) Triple labeling for PDGFR-β, nestin and collagen IV showing that expression of the three proteins generally overlap in the lesion core. The boxed area in (O) is enlarged in (Q–T) . (P) Histogram of the intensity profiles of PDGFR-β, nestin and collagen IV along the indicated area (white arrows in Q–S ) showing that the three signals share overlapping profiles. Cell nuclei are stained with DAPI. Scale bars = 100 μm for (A,B,O) ; 20 μm for (I–N„Q–T) ; 10 μm for (C–H) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Injection, Labeling, Staining, Expressing

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Ultrastructural characterization of PDGFR-β/nestin double-positive cells in the lesion core. Confocal microscopic image of a semi-thin section double-labeled with PDGFR-β and nestin (A,F) , the corresponding transmission electron microscopic image (C,H) , and the overlay image of confocal microscopic data and the corresponding electron microscopic image (B,G) obtained from days 7 (A–E) and 14 (F–K) post-lesion. (D,E,L–K) Higher-magnification views of the boxed areas in (B,C,G,H) , respectively. PDGFR-β/nestin double-positive cells have euchromatic nuclei and rER with dilated cisternae (#). Note that at 7 days, the cytoplasmic processes of these cells are still found extending around the vascular wall on the abluminal side of a smooth muscle (SM) cell and endothelial cells (EN), while at 14 days, they exhibit slender cytoplasmic processes branching out into the extravascular spaces. Cell nuclei are stained with DAPI. Scale bars = 2 μm for (A,B) , and (F–H) ; 1 μm for (C–E,K) ; 0.5 μm for (J) ; 0.2 μm for (I) .

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Labeling, Transmission Assay, Staining

Journal: Frontiers in Molecular Neuroscience

Article Title: PDGFR-β-Positive Perivascular Adventitial Cells Expressing Nestin Contribute to Fibrotic Scar Formation in the Striatum of 3-NP Intoxicated Rats

doi: 10.3389/fnmol.2018.00402

Figure Lengend Snippet: Schematic representation of dynamic PDGFR-β-positive cells in the lesion core in striata treated with 3-NP. (A) In the saline-treated control striatum, PDGFR-β-positive cells with thin processes are associated with larger caliber vessels having one or two layers of smooth muscle cells. These cells are located outside the smooth muscle cells and are surrounded by the glia limitans of astroglial processes. (B) At 3–7 days post-lesion, PDGFR-β-positive cells are highly proliferative and express nestin and vimentin. They have large euchromatic nuclei and dilated cisternae of rER, indicating the presence of active collagen synthesis. They also migrated to the adjacent microvascular wall and wrap around or directly abut the abluminal surface of endothelial cells or pericytes, despite the ongoing association with the vasculature. (C) At 14–28 days, the lesion core is progressively infiltrated by migrating PDGFR-β-positive cells expressing nestin and vimentin. They have highly branched cytoplasmic processes that are frequently in close apposition or are even interwoven with each other, forming a network. In addition, PDGFR-β-positive somata and processes have close apposition with brain macrophages, nearly always at the branch points of distal processes.

Article Snippet: Primary antibodies and dilutions were as follows: rabbit monoclonal antibody against PDGFR-β (1:200; Abcam), mouse monoclonal antibody against RECA1 (1:200; Bio-Rad, Hercules, CA, USA), chicken polyclonal antibody against glial fibrillary acidic protein (GFAP; 1:500; Millipore), goat polyclonal antibody against type IV collagen (1:100; Bio-Rad), mouse monoclonal antibody against nestin (1:500; Bio-Rad), goat polyclonal antibody against ionized calcium-binding adaptor molecule 1 (Iba1; 1:500; Abcam), chicken polyclonal antibody against vimentin (1:500, Millipore), mouse monoclonal antibody to NG2 (1:500; Millipore), or rabbit polyclonal antibody against Ki-67 (1:1,000; Leica Biosystems, Wetzlar, Germany).

Techniques: Saline, Control, Expressing

Journal: The Journal of Neuroscience

Article Title: A Crucial Role for Primary Cilia in Cortical Morphogenesis

doi: 10.1523/JNEUROSCI.2084-08.2008

Figure Lengend Snippet: Primary cilia project into the ventricle of embryonic forebrain. A–I, TEM (A–G) and (H, I) scanning EM of cilia projecting into the dorsolateral telencephalic ventricles (V) of E12.5 wild-type (A–D, H) and cbs/cbs (E–G, I) embryos. A, E, Cilium cut longitudinally. B–D, F, G, Cross sections of ventricular cilia, revealing the basal body (B, F), characteristic “9 + 0” ciliary morphology (C, G), and tapering to a “2 + 0” structure (D). E, Proximal-to-distal tapering can clearly be seen (arrow). H, I, Arrows indicate cilia. J–L, Immunofluorescence analysis of Ift88 expression in dorsolateral telencephalon of E11.5 Ift88Δ2–3βgal embryos, using an antibody recognizing β-galactosidase (J, L, red; K, green), which is expressed from the Ift88 locus. The ventricular (V) and pial (P) surface is at bottom (J, K), and top (L) of the panels, respectively. Arrows indicate characteristic somatic β-galactosidase deposits in cells colabeled with the following markers: Nestin- (J, green) and RC2-positive (K, red)VZ cells and newborn neurons (L, green, TuJ1 antibody). J–L, Blue, DAPI-labeled nuclei. Scale bars: A, C, E, G, 200 nm; B, D, F, 100 nm; H, I, 1 μm; J, K, L, 10 μm.

Article Snippet: Primary antibodies (Ab): rabbit anti-β-galactosidase (clone 55976, ICN/Cappel) 1:2000, mouse anti-nestin (Rat 401 clone; BD PharMingen) 1:500, RC2 (DSHB) 1:10, mouse anti-β tubulin III (TuJ1 clone; Covance) 1:1500, rabbit anti-phospho-histone H3 (Ser10, rabbit 06–570; Upstate) 1:200, and rabbit anti-Pax6 (AB5409, Millipore) 1:3000.

Techniques: Immunofluorescence, Expressing, Labeling

Journal: The Journal of Neuroscience

Article Title: A Crucial Role for Primary Cilia in Cortical Morphogenesis

doi: 10.1523/JNEUROSCI.2084-08.2008

Figure Lengend Snippet: The cbs mutant exhibits a pronounced disorganization of the dorsal telencephalon. A–D, H, Hematoxylin-stained coronal sections of E12.5 wild-type (A, C) and cbs/cbs (B, D, H–J) embryos. A, B, E-G, Rostral and (C, D, H–J) caudal telencephalon. E–G, Hematoxylin-stained coronal sections of a E11.5 Ift88ko/− embryo (E), a Ift88ko/cbs compound heterozygote littermate (F), and a cbs/cbs embryo (G). A, C, E, Inset (lower left) indicates the plane of section for (A, B), (C, D), and (E–G), respectively. H, Caudal-most telencephalon showing lateral heterotopias (left, boxed). Enlargement of the boxed region shows them to have a rosette-like morphology (right). B, D, G, H, Arrows indicate heterotopias. G, Arrowhead indicates VZ. I–L, Mitotic cells revealed with an anti-phosphorylated-histone H3 antibody (green, PH3) in subpial heterotopias (I, J) and VZ of the dorsolateral cortex (K) and the ganglionic eminences (L) in E12.5 wild-type and cbs/cbs embryos. Red = anti-nestin antibody. I–L, Arrows indicate nestin/PH3-double positive cells. Arrowheads indicate sub-VZ mitoses. Asterisks indicate blood cells. M, N, O, Quantitation of PH3 staining in the cortex (cortex) and ganglionic eminences (GE), expressed as the number of PH3-positive (M) and double nestin/PH3-positive (N) cells per 100 μm of VZ, and in the number of basally located (i.e., >30 μm from the ventricular zone) PH3-positive cells per 1 mm2 (O). ***p < 0.001, Student's t test. A–L, Dorsal is to the top. H–L, Lateral is to the left; V, ventricle; P, pial surface. Scale bars: A–G, H (left), 300 μm; H (right), I–L, 50 μm.

Article Snippet: Primary antibodies (Ab): rabbit anti-β-galactosidase (clone 55976, ICN/Cappel) 1:2000, mouse anti-nestin (Rat 401 clone; BD PharMingen) 1:500, RC2 (DSHB) 1:10, mouse anti-β tubulin III (TuJ1 clone; Covance) 1:1500, rabbit anti-phospho-histone H3 (Ser10, rabbit 06–570; Upstate) 1:200, and rabbit anti-Pax6 (AB5409, Millipore) 1:3000.

Techniques: Mutagenesis, Staining, Quantitation Assay

Journal: Cells

Article Title: Analyzing Sex-Specific Dimorphism in Human Skeletal Stem Cells

doi: 10.3390/cells12232683

Figure Lengend Snippet: SSCs (p2-3) expressed stem cell markers and lacked neural crest markers. ( A ) SSCs derived from female donor 2 expressed CD164, Nestin, CD133, and SOX9 and lacked the expression of SLUG and p75. ( B ) Male donor 14-derived SSCs expressed CD164, Nestin, CD133, and SOX9 but did not express SLUG or p75. ( C ) Semiquantitative analysis showed high Nestin (65.6–89.4%), CD133 (43.5–96.3%), CD164 (82.7–91.0%), and SOX9 (98.1–99.3%) expression in three female SSC populations but lacking p75 (0.0–0.0%) and SLUG (1.1–2.5%) expression. ( D ) Semiquantitative analysis of three male SSC populations unraveled high levels of Nestin (55.4–88.2%), CD133 (50.9–78.8%), CD164 (62.8–75.8%), and SOX9 (97.1–99.0%), but p75 (0.0–0.0%) and SLUG (1.1–3.2%) expressions were deficient.

Article Snippet: Primary antibodies against p65 (rabbit, 1:400, Cell Signaling Technology, Danvers, MA, USA, mAb#8242), Nestin (mouse,1:200, Merck Millipore, Burlington, MA, USA, MAB353), CD164 (mouse, 1:100, Santa Cruz Biotechnology, Dallas, TX, USA, sc-271179), CD133 (rabbit, 1:100, Novus Biologicals, Centennial, CO, USA, NB120-16518,), SLUG (rabbit, 1:400, Cell Signaling Technology, C1967), SOX9 (rabbit,1:400, Invitrogen, Waltham, MA, USA, #702016,), p75 (mouse, 1:100, Sigma Aldrich, N5408), and β-actin (rabbit, 1:200, Cell Signaling Technology, #4970) were diluted in 1× PBS and incubated for 1 h at room temperature.

Techniques: Derivative Assay, Expressing