Journal: bioRxiv

Article Title: GATA2 controls lymphatic endothelial cell junctional integrity and lymphovenous valve morphogenesis through miR-126

doi: 10.1101/660068

Figure Lengend Snippet: E12.0 Wild type and Lyve1-Cre;Gata2 f/f littermates or Tg(Prox1-tdTomato) and Tg(Prox1-tdTomato); Lyve1-Cre;Gata2 f/f littermates were frontally sectioned and IHC was performed for LVV-EC markers (PROX1, tdTomato, FOXC2, ITGA5, ITGA9), ECM components (FN1, LAMA5), and cell junction proteins (CD31, VE-Cadherin, GJA4, CLDN5). No obvious differences were observed between control and mutant samples. Abbreviations: A, artery; LS, lymph sac. Measuring bar: (A-P) 100 µm. Statistics: n=3 embryos and 6 LVV complexes per genotype per antibody.

Article Snippet: Primary antibodies for immunohistochemistry: rabbit anti-PROX1 (Cat# 11-002, Angiobio, San Diego, CA, USA), goat anti-human PROX1 (Cat# AF2727, R&D Systems, Minneapolis, MN, USA), sheep anti-mouse FOXC2 (Cat# AF6989, R&D Systems, Minneapolis, MN, USA), goat anti-mouse VEGRF3 (Cat# AF743, R&D Systems, Minneapolis, MN, USA), rat anti-mouse CD31 (Cat# 553370, BD Pharmingen, San Jose, CA, USA), goat anti-mouse ITGA9 (Cat # AF3827, R&D Systems, Minneapolis, MN, USA), rat anti-mouse VE-Cadherin (Cat# 550548, BD Pharmingen, San Jose, CA, USA), hamster anti-mouse PDPN (Cat# 127401, Biolegend, San Diego, CA, USA), rat anti-mouse ITGA5 (Cat #553319, BD Pharmingen, San Jose, CA, USA), goat anti-mouse GATA2 (Cat #AF2046, R&D Systems, Minneapolis, MN, USA), rabbit anti-mouse CX37 (Cat #40-4200, Life technologies, Grand Island, NY, USA), rabbit anti-mouse LAMA5 (Abcam, Cambridge, MA, USA), rabbit anti-human Fibronectin (Cat# ab2413, Abcam, Cambridge, MA, USA), Goat anti-human ANGPT2 (Cat# AF623, R&D Systems, Minneapolis, MN, USA), goat anti-mouse EGFL7 (Cat # AF3089, R&D Systems, Minneapolis, MN, USA), rabbit anti-mouse CLDN5 (Cat #34-1600, Thermo Fisher Scientific, Rockford, IL, USA), rabbit anti-mouse LYVE-1 (Cat # 11-034, Angiobio, San Diego, CA, USA).

Techniques: Mutagenesis

Journal: The Journal of comparative neurology

Article Title: Domain-specific distribution of gap junctions defines cellular coupling to establish a vascular relay in the retina

doi: 10.1002/cne.24699

Figure Lengend Snippet: Primary antibodies

Article Snippet: All primary antibodies are listed in . table ft1 table-wrap mode="anchored" t5 Table 1. caption a7 Molecular marker Immunogen Host Dilution Clone Source RRID Albumin whole mouse albumin goat 1:800 poly Bethyl, A90–234A RRID:AB_67122 claudin5 clone 4C3C2, Alexa488 Synthetic peptide from the mouse claudin5 mouse 1:10000 mono Invitrogen, 352588 RRID:AB_2532189 Connexin 43 (Cx43s) PSSRASSRASSRPRPDDLEI rabbit 1:2000 poly Sigma, C6219 RRID:AB_476857 Connexin 43 (Cx43a) (C)HAQPFDFPDDNQNSK rabbit 1:10000 poly Alomone, ACC-201 RRID:AB_10917597 Connexin 40 (C)GHRFPQGYHSDKR rabbit 1:3000 poly Alomone, ACC-205 RRID:AB_2340916 Connexin 37 (C)EHQMAKISVAEDGR rabbit 1:2000 poly Alomone, ACC-204 RRID:AB_2722598 Smooth muscle actin, SMA, 1A4 N-terminal synthetic decapeptide of α-SMA mouse 1:1000 mono Sigma, A5228 RRID:AB_262054 Glial fibrillary acidic protein, GFAP purified bovine GFAP chicken 1:3000 poly Millipore, AB5541 RRID:AB_177521 CD31/PECAM-1 Mouse myeloma cell NS0-derived recombinant mouse CD31 Glu18-Lys590 goat 1:6000 poly RD, AF3628 RRID:AB_2161028 Open in a separate window Primary antibodies

Techniques: Marker, Purification, Recombinant

Journal: Genes & Development

Article Title: Mechanotransduction activates canonical Wnt/β-catenin signaling to promote lymphatic vascular patterning and the development of lymphatic and lymphovenous valves

doi: 10.1101/gad.282400.116

Figure Lengend Snippet: Wnt/β-catenin signaling is necessary and sufficient to regulate the expression of VEC markers in LECs. ( A – D ) Primary human LECs were cultured in the presence or absence of OSS for 48 h. Subsequently, cells were analyzed by IHC for the indicated markers ( A , B ), Western blot ( C ), or quantitative PCR (qPCR) ( D ). ( A , B ) IHC revealed the up-regulation of FOXC2 and β-catenin expression by OSS. ( C ) Western blot revealed an increase in the expression levels of total and active β-catenin. Valve-expressed transcription factors GATA2 and FOXC2 are also increased. PROX1 levels are not obviously changed. ( D ) qPCR validated the up-regulation of FOXC2. Additionally, target genes of the Wnt/β-catenin signaling pathway—Axin2, Cyclin-D1, and c-Jun—are increased. ( E ) LECs were cultured under static or OSS conditions in the presence or absence of 25 µM iCRT3, an antagonist of Wnt/β-catenin signaling, for 48 h. Western blot revealed a modest down-regulation of FOXC2, GATA2, and PROX1 expression by iCRT3 under static conditions. In contrast, iCRT3 dramatically inhibited the OSS-mediated up-regulation of FOXC2 and GATA2 expressions. The numbers in red indicate the relative expression of FOXC2 as measured densitometrically. ( F – I ) Primary human LECs were cultured in the presence of 0.5 µM Bio, an agonist of the Wnt/β-catenin signaling pathway, for 12 h. Subsequently, cells were analyzed by IHC for the indicated markers ( F , G ), Western blot ( H ), or qPCR ( I ). The results show that Bio enhances the expression of valve markers FOXC2, GATA2, PROX1, and CX37. Bars: A , B , F , G , 100 µm. n = 3 for each experiment. (*) P < 0.05; (**) P < 0.01; (***) P < 0.001.

Article Snippet: Primary antibodies used for immunostaining were as follows: rabbit anti-PROX1 (AngioBio), goat anti-human PROX1 (R&D Systems), sheep anti-mouse FOXC2 (R&D Systems), goat anti-mouse VEGFR3 (R&D Systems), goat anti-mouse LYVE1 (R&D Systems), rat anti-mouse CD31 (BD Pharmingen), goat anti-mouse GATA2 (R&D Systems), rabbit anti-total β-catenin and rabbit anti-active β-catenin antibodies (both from Cell Signaling Technologies), goat anti-mouse NRP2 (R&D Systems), chicken anti-GFP (Abcam), rat anti-mouse Endomucin (eBioscience), rabbit anti-CX37 (Invitrogen), and Cy3-conjugated monoclonal anti-α-SMA (Sigma-Aldrich).

Techniques: Expressing, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: (A through D) Histologic sections of kidney cortex from a mouse kept on normal-salt diet stained for Cx40 (A), Cx37 (B), Cx43 (C), Cx45 (D) together with renin (green color). Bar = 50 μm. Circles highlight glomerulus (gl); egm, extraglomerular mesangium; aa, afferent arteriole; arrowheads highlight renin-expressing cells; arrows indicate endothelial Cx staining; ▵, Cx45 staining in smooth muscle cells. Cx37, Cx40, and Cx43 are found in association with renin-expressing cells and with endothelial cells of the aa. Whereas Cx40 and Cx43 immunoreactivity extends more evenly over the whole glomeruli, Cx37 immunoreactivity is more focused to the entrance of the aa into the capillary network. Cx45 immunoreactivity is not associated with renin-expressing cells but clearly is seen in VSMCs of aa and in intraglomerular cells.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Staining, Expressing

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: (A through D) Histologic sections of kidney cortex from a mouse kept on low-salt diet in combination with the ACEI enalapril (10 mg/kg per d), stained for Cx40 (A), Cx37 (B), Cx43 (C), and Cx45 (D) together with renin (green color). Bar = 50 μm. Circles highlight gl; arrowheads highlight renin-expressing cells; arrows indicate renin immunoreactivity without immunoreactivity of the respective Cx. Treatment of the mice led to an upstream recruitment of renin-expressing cells along the aa. All renin positive cells also stained for Cx40, whereas Cx37 immunoreactivity was absent from renin-expressing cells but still present in the endothelium of aa. Cx43 immunoreactivity was associated with renin-expressing cells close to the glomerular vascular poles but faded in renin-expressing cells with increasing distance to the vascular pole. Cx45 immunoreactivity did not co-localize with renin.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Staining, Expressing

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Renin, Cx37, Cx40, Cx43, and Cx45 mRNA abundance in kidney cortex of mice treated with a combination of low-salt diet (0.02% wt/wt) with the ACEI enalapril (10 mg/kg per d; ls/enal), with enalapril (10 mg/kg per d) alone (enal), with low-salt diet (0.02% wt/wt; ls), with normal-salt diet (0.6% wt/wt), or with high-salt diet (4% wt/wt; hs). Data are means ± SEM of five mice in each group. *P < 0.05 versus vehicle-treated mice on normal-salt diet.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques:

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Histologic sections of kidney cortex from a normal 17-d-old mouse fetus. (A) Immunostaining for Cx43 alone. (B) Co-immunostaining for renin (green) and Cx43 (red). (C) Co-immunostaining for renin (green), α-SMA (blue), and Cx43 (red). (D) Immunostaining for Cx45 alone. (E) Co-immunostaining for renin (green) and Cx45 (red). (F) Co-immunostaining for renin (green), α-SMA (blue), and Cx45 (red). Bar = 50 μm. Arrowheads indicate regions of renin expression without Cx staining; arrows indicate endothelial Cx staining; lines, Cx45 co-localizing with renin; *, Cx45 in smooth muscle cells with low Cx45 staining.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Immunostaining, Expressing, Staining

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Histologic sections of kidney cortex from a Cx40-deficient mouse kept on a normal-salt diet. (A) Immunostaining for Cx40 alone. (B) Co-immunostaining for renin (green), Cx40 (red), and α-SMA (blue). (C) Immunostaining for Cx37 alone. (D) Co-immunostaining for renin (green), Cx37 (red), and α-SMA (blue). (E) Immunostaining for Cx43 alone. (F) Co-immunostaining for renin (green), Cx43 (red), and α-SMA (blue). Bar = 50 μm. Circles highlight gl; arrowheads indicate renin-expressing cells; arrows indicate endothelial Cx staining; ▵, staining for Cx43 in venous endothelium.

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Immunostaining, Expressing, Staining

Journal:

Article Title: Connexin Expression in Renin-Producing Cells

doi: 10.1681/ASN.2008030252

Figure Lengend Snippet: Primer sequences used for cDNA amplification

Article Snippet: Antibodies used for immunostaining of Cxs were purchased from commercial providers: Anti-Cx40 (goat anti-mouse; Santa Cruz Biotechnology, Santa Cruz, CA), anti-Cx37 (rabbit anti-mouse; Alpha Diagnostic Int., San Antonio, TX), anti-Cx43 (rabbit anti-mouse; Sigma, St. Louis, MO, and Zymed, Basel, Switzerland), anti-Cx45 (rabbit anti-mouse 32 ), and anti–α-SMA (mouse anti-mouse; Beckman Coulter Immunotech, Marseille, France).

Techniques: Sequencing