Journal: Physiological Reports

Article Title: Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis

doi: 10.14814/phy2.13318

Figure Lengend Snippet: Differentiation of neural stem cells into neurons in the Matrigel culture. After seeding the hippocampus fragments in collagen gels under an ALI wells and cultured for 7 days, they were passaged into Matrigel and cultured for 7 days (A). Bright field pictures were taken at day 0 and 7 post‐passage (B). Scale bar: 200 μm. Expression of Nestin (C), DCX (D), a neuronal cell marker, MAP2ab (E), a granule cell marker, Prox‐1 (F) and an astrocytes marker, GFAP (G). Representative immunofluorescence pictures are shown ( n = 4). Scale bar: 50 μm.

Article Snippet: Antibody sources were as follows: Neuronal nuclei (NeuN) (Cell Signaling Technology, Inc., Danvers, MA); Nestin (Immuno‐Biological Laboratories, Gunma, Japan); Doublecortin (DCX) (Sigma‐Aldrich); ΜAP2ab, Prox‐1 (Bioss antibodies, Woburn, MA); Tuj1 (Bethyl Laboratories, Montgomery, TX); Glial Fibrillary Acidic Protein (GFAP) (Diagnostic BioSystems, Pleasanton, CA); Valosin‐containing protein (VCP) (GeneTex, Inc., Irvine, CA).

Techniques: Cell Culture, Expressing, Marker, Immunofluorescence

Journal: Physiological Reports

Article Title: Establishment of a novel three‐dimensional primary culture model for hippocampal neurogenesis

doi: 10.14814/phy2.13318

Figure Lengend Snippet: Expression changes of neuronal differentiation marker proteins in the Matrigel culture. After the hippocampus tissues were cultured in ALI wells and seeded into Matrigel, expression of Nestin (A, n = 4), DCX (B, n = 5), a neuronal cell marker, Tuj1 (C, n = 3) and Prox‐1 (D, n = 4) was determined by Western blotting at day 0 and 7 post‐passaging. Equal protein loading was confirmed using total VCP antibody. * P < 0.05 versus P1 Day 0.

Article Snippet: Antibody sources were as follows: Neuronal nuclei (NeuN) (Cell Signaling Technology, Inc., Danvers, MA); Nestin (Immuno‐Biological Laboratories, Gunma, Japan); Doublecortin (DCX) (Sigma‐Aldrich); ΜAP2ab, Prox‐1 (Bioss antibodies, Woburn, MA); Tuj1 (Bethyl Laboratories, Montgomery, TX); Glial Fibrillary Acidic Protein (GFAP) (Diagnostic BioSystems, Pleasanton, CA); Valosin‐containing protein (VCP) (GeneTex, Inc., Irvine, CA).

Techniques: Expressing, Marker, Cell Culture, Western Blot, Passaging

Journal: International Journal of Inflammation

Article Title: Interleukin-1 β Expression Is Required for Lysophosphatidic Acid-Induced Lymphangiogenesis in Human Umbilical Vein Endothelial Cells

doi: 10.4061/2011/351010

Figure Lengend Snippet: Lysophosphatidic acid- (LPA-) induced lymphatic marker expressions are mediated through endothelial growth factor receptor (EGFR) transactivation-, matrix metalloproteinase- (MMP-), LPA1/3-, and interleukin- (IL-) 1R-dependent pathways in human umbilical vein endothelial cells (HUVECs). Starved HUVECs were pretreated with AG1478 (100 nM), AF12198 (10 nM), GM6001 (10 μ M), Ki16425 (10 μ M), and toxin B (10 nM) for 1 h, followed by LPA (5 μ M) treatment for an additional 8 h. HUVECs were dissociated by trypsinization and fixed with 4% paraformaldehyde. Fixed cells were labeled with antibodies for Prox-1, LYVE-1, and podoplanin, and then analyzed by CyFlow. Cells were also stained with a normal mouse or goat immunoglobulin G antibody, which served as a negative control.

Article Snippet: The Matrigel was fixed, blocked, permeabilized, and stained with rabbit anti-mouse Prox-1 (Abcam), followed by incubation with a goat anti-rabbit AlexaFluor-555-conjugated secondary antibody (Molecular Probes).

Techniques: Marker, Labeling, Staining, Negative Control

Journal: International Journal of Inflammation

Article Title: Interleukin-1 β Expression Is Required for Lysophosphatidic Acid-Induced Lymphangiogenesis in Human Umbilical Vein Endothelial Cells

doi: 10.4061/2011/351010

Figure Lengend Snippet: Lysophosphatidic acid- (LPA-) stimulated endothelial cell tube formation in vitro is mediated through endothelial growth factor receptor (EGFR) transactivation-, matrix metalloproteinase- (MMP-), LPA1/3-, and interleukin- (IL-) 1R-dependent mechanisms. Starved HUVECs were pretreated with AG1478 (100 nM), AF12198 (10 nM), GM6001 (10 μ M), Ki16425 (10 μ M), and toxin B (10 nM) for 1 h, followed by LPA (5 μ M) treatment for an additional 24 h, and then cells were seeded onto Matrigel-coated plates. After 6 h of plating, Matrigel of each experiment was fixed and subjected to an immunocytochemical assay. The Matrigel was stained with PECAM-1 or a Prox-1 primary antibody followed by an FITC-conjugated secondary antibody. Images were taken after plating and visualized by immunofluorescence microscopy.

Article Snippet: The Matrigel was fixed, blocked, permeabilized, and stained with rabbit anti-mouse Prox-1 (Abcam), followed by incubation with a goat anti-rabbit AlexaFluor-555-conjugated secondary antibody (Molecular Probes).

Techniques: In Vitro, Staining, Immunofluorescence, Microscopy

Journal: Scientific Reports

Article Title: New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury

doi: 10.1038/s41598-021-89616-3

Figure Lengend Snippet: Immunohistochemical staining images showing PROX1 expression in ( A ) normal mouse brain tissue and ( B – F ) mouse brain tissue on days 3, 7, 15, 21 and 30 days, following pTBI. ( A ) PROX1 is not expressed in normal mouse brain tissue. PROX1 expression (brown; black arrows) was detected around the injury site (red arrow) on days ( B ) 3 and ( C ) 7. ( D ) PROX1-expressing cells and tubular structures (black arrows) were observed around the injury site on day 15 and a small number of black hemosiderin particles (black arrowheads) were visible. ( E , F ) PROX1 expression (black arrows) and black hemosiderin particles (black arrowheads) were still visible at the injury site on days 21 and 30. ( G ) Immunohistochemical results of PROX1 were analyzed by Image-Pro Plus 6.0 software. The average density (IOD) of PROX1-positive cells in normal mouse brain tissue and in mouse brain tissue on days 3, 7, 15, 21 and 30 days, respectively, following pTBI. The n = 10/group was used for calculating the average IOD. Comparisons between groups were performed using the Kruskal–Wallis H test followed by Bonferroni post-hoc analysis.

Article Snippet: The sections were first incubated with rabbit anti-mouse PROX1 antibody (1:100; Boster Biological Technology Co., Wuhan, China), rabbit anti-mouse CD34 antibody (1:100; BosterBiological Technology) or goat anti-mouse LYVE-1 antibody (1:100; R&D Systems, Shanghai, China) at 4 °C for 24 h. Next, the sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG secondary antibodies.

Techniques: Immunohistochemical staining, Staining, Expressing, Software

Journal: Scientific Reports

Article Title: New lymphatic cell formation is associated with damaged brain tissue clearance after penetrating traumatic brain injury

doi: 10.1038/s41598-021-89616-3

Figure Lengend Snippet: Double immunofluorescence staining images showing LYVE-1/PROX1 expression in brain tissue 7 days after pTBI. Expression of ( A ) LYVE-1 (green); ( B ) PROX1 (red); and ( C ) merge (yellow).

Article Snippet: The sections were first incubated with rabbit anti-mouse PROX1 antibody (1:100; Boster Biological Technology Co., Wuhan, China), rabbit anti-mouse CD34 antibody (1:100; BosterBiological Technology) or goat anti-mouse LYVE-1 antibody (1:100; R&D Systems, Shanghai, China) at 4 °C for 24 h. Next, the sections were incubated with horseradish peroxidase-labeled goat anti-rabbit IgG or rabbit anti-goat IgG secondary antibodies.

Techniques: Double Immunofluorescence Staining, Expressing