rabbit anti-zo-1 (mid)  (Thermo Fisher)

Journal: BMC Cancer

Article Title: Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes

doi: 10.1186/1471-2407-9-229

Figure Lengend Snippet: Knockdown of p27 disrupts VHL-mediated tight junction formation . (A) RCC10 cells stably expressing WT pVHL 30 were infected with retroviruses containing shRNA constructs directed at luciferase as control (sh Luc) or p27 (sh p27). Approximately 2 weeks post-infection, cell lysates were equally loaded and subjected to p27 and α-tubulin western blotting. (B) RCC10 WT VHL cells infected with shRNA to luciferase or p27 were grown to confluence on collagen I and photographed by digital phase microscopy (original magnification of 100×). (C) RCC10 WT VHL cells infected with shRNA to luciferase or p27 were grown to confluence on coverslips and immunostaining for ZO-1 (left panels) was performed (original magnification of 1000×). DAPI labeled nuclei of corresponding cells are also shown (right panels). (D and E) 786-O cells stably expressing pVHL 19 were infected and analyzed as in (A) and (C).

Article Snippet: Rabbit anti-ZO-1 (Mid) was from Zymed.

Techniques: Stable Transfection, Expressing, Infection, shRNA, Construct, Luciferase, Western Blot, Microscopy, Immunostaining, Labeling

Journal: BMC Cancer

Article Title: Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes

doi: 10.1186/1471-2407-9-229

Figure Lengend Snippet: Contrasting regulation of tight junctions among VHL mutants of different disease types in RCC10 . RCC10 cells lines (as indicated; all created by retroviral infection) were grown to confluence on coverslips and immunostaining for ZO-1 (left panels) was performed (original magnification of 1000×). DAPI labeled nuclei of corresponding cells are also shown (right panels).

Article Snippet: Rabbit anti-ZO-1 (Mid) was from Zymed.

Techniques: Infection, Immunostaining, Labeling

Journal: BMC Cancer

Article Title: Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes

doi: 10.1186/1471-2407-9-229

Figure Lengend Snippet: Contrasting levels of ZO-1 fragments among VHL mutants of different disease types in RCC10 . RCC10 cells lines (as indicated; all created by retroviral infection) were grown to confluence. Cell lysates were prepared and equally loaded and separated by SDS-PAGE. Western blots were performed for ZO-1 and α-tubulin. Arrows indicate ZO-1 immunoreactive proteins.

Article Snippet: Rabbit anti-ZO-1 (Mid) was from Zymed.

Techniques: Infection, SDS Page, Western Blot

Journal: BMC Cancer

Article Title: Differences in regulation of tight junctions and cell morphology between VHL mutations from disease subtypes

doi: 10.1186/1471-2407-9-229

Figure Lengend Snippet: Tight junctions are affected by levels of HIF-2α in 786-O . (A) Parental 786-O cells (786-O), 786-O cells stably transfected with VHL (VHL +), and 786-O cells infected with an empty retrovirus (pSuperRetro) or infected with HIF-2α shRNAs (HIF2α shRNA), as described in , were grown for 1 week past confluence on collagen I coated culture dishes. Cell lysates were prepared, equally loaded, and western blotted for HIF-2α, cyclin D1, p27, α-tubulin and ZO-1 (both upper and lower immunoreactive species that were seen in Figure 9). (B) Indicated cell lines that were assayed in (A) were grown to confluence on coverslips and immunostained for ZO-1 (left panels; original magnification of 1000×). DAPI labeled nuclei of corresponding cells are also shown (right panels). (C) Indicated cell lines that were assayed in (A) and (B) were grown to confluence on collagen I and photographed by digital phase microscopy (original magnification of 100×).

Article Snippet: Rabbit anti-ZO-1 (Mid) was from Zymed.

Techniques: Stable Transfection, Transfection, Infection, shRNA, Western Blot, Labeling, Microscopy

Journal: PLoS ONE

Article Title: Defects in the Outer Limiting Membrane Are Associated with Rosette Development in the Nrl −/− Retina

doi: 10.1371/journal.pone.0032484

Figure Lengend Snippet: To analyze the integrity of the OLM in the Nrl −/− retina, sections were stained with antibodies specific for Zo-1 and beta-catenin (red), components of the OLM junctions, and S-opsin (green) to label OSs. A. In the WT retina, the Zo-1 staining forms a straight continuous line. In the Nrl −/− retina, gaps in OLM staining (arrows) can be seen that correspond spatially and temporally with the appearance of nascent rosettes and aberrant photoreceptors (arrowheads). B. Beta-catenin staining can be seen at the OLM as well as in the OPL and INL. In the Nrl −/− retina, gaps in OLM staining (arrows) can be seen that correspond with the appearance of nascent rosettes and aberrant photoreceptors (arrowheads). OLM: outer limiting membrane, ONL: outer nuclear layer, INL: inner nuclear layer. Scale bar, 20 µm.

Article Snippet: Primary antibodies were used as follows: rabbit anti-S-opsin (generated in-house and characterized previously ) diluted at 1∶1000; goat anti-S-opsin (H-17, Santa Cruz Biotechnology, Santa Cruz, CA) diluted at 1∶500; rabbit anti-recoverin (generously shared by Dr. James F. McGinnis, OUHSC ) diluted at 1∶5000; mouse monoclonal anti-BrdU (G3G4, the monoclonal antibody developed by Stephen J. Kaufman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biology, Iowa City, IA) diluted at 1∶10; rabbit anti-B-catenin (C2206, Sigma-Aldrich, St. Louis, MO), diluted at 1∶1000; rabbit anti-ZO-1(Mid) (40–2200, Invitrogen, Carlsbad, CA) diluted at 1∶100; mouse monoclonal anti-ezrin (3C12, Abcam, Cambridge, MA) diluted at 1∶50; rabbit anti-mCAR-LUMIj (generously shared by Dr. Cheryl Craft, USC , , , ); rabbit anti-water channel aquaporin 4 (A5971, Sigma-Aldrich, St. Louis, MO) and rabbit anti-RDS-CT (generated in-house and characterized previously ) diluted at 1∶2000.

Techniques: Staining

Journal: Cell and Tissue Research

Article Title: The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

doi: 10.1007/s00441-014-1969-7

Figure Lengend Snippet: Agrin 000 and agrin 748 increase junctional localization of vascular endothelial cadherin ( VE-cadherin ), β-catenin, and zonula occludens-1 ( ZO-1 ) in bEnd5 cells. Immunofluorescence (IF) staining was performed after the culturing of bEnd5 cells for 48 h in the absence or presence of chicken agrin 000 or agrin 748. Cells were stained for VE-cadherin ( a , a’ , a’’ ), β-catenin ( b , b’ b’’ ), ZO-1 ( c , c’ , c’’ ), ZO-1-associated Y-box factor (ZONAB; d , d’ , d’’ ), and F-actin ( e , e’ , e’’ ). Representative micrographs from 4–5 independent experiments. Bar 50 μm. To quantify the IF signal at the junctions, the gray values of the VE-cadherin ( f ), β-catenin ( g ), ZO-1 ( h ), and ZONAB ( i ) IF signals at the bEnd5 junctions were measured with ImageJ software. Symbols represent the mean values of each independent experiment ( f , g , n = 5; h , n = 6; i , n = 4); the means over all experiments is represented by the horizontal lines . Gray values were normalized to the control condition for every independent experiment ( n.s. not significant)

Article Snippet: The following primary antibodies were used for immunohistochemistry (IHC) and immunofluorescence (IF) staining: monoclonal mouse anti-chicken agrin antibodies 5B1 (1:500) and C3 (1:200; Reist et al. ), polyclonal rabbit anti-mouse agrin serum 204 (1:1000; Eusebio et al. ), polyclonal rabbit anti-claudin-5 (1:100; 34–1,600, Zymed), polyclonal rabbit anti-ZO-1 (1:100; 61–7,300, Invitrogen), polyclonal rabbit anti-ZO-1-associated Y-box factor (ZONAB, Mid; 1:150; 40–2,800, Invitrogen), polyclonal rabbit anti-ZO-2 (1:100; 71–1,400, Zymed), polyclonal rabbit anti-occludin (1:50; 71–1,500, Zymed), polyclonal rabbit anti-β-catenin (1:2000; C2206, Sigma), monoclonal rat anti-VE-cadherin (11D4.1, hybridoma supernatant; Gotsch et al. ; kindly provided by Prof. Dietmar Vestweber, Münster, Germany), monoclonal rat anti-JAM-A (BV12; Bazzoni et al. ; kindly provided by Prof. Elisabetta Dejana, Milan, Italy), polyclonal rabbit anti-laminin (1:1000 for IHC, 1:4000 for IF; Z0097, DakoCytomation), polyclonal rabbit anti-caveolin (1:450; 610,059, BD Transduction Laboratories), biotinylated polyclonal goat anti-mouse IgG (1:200; BA9200, Vector).

Techniques: Immunofluorescence, Staining, Software

Journal: Cell and Tissue Research

Article Title: The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

doi: 10.1007/s00441-014-1969-7

Figure Lengend Snippet: Agrin 000 and agrin 748 do not increase expression levels of VE-cadherin, β-catenin and ZO-1 in bEnd5 cells. a , c , e Quantitative reverse transcription plus the polymerase chain reaction was performed to analyze VE-cadherin, β-catenin, and ZO-1 mRNA levels in bEnd5 cells cultured for 24 h on agrin 000, agrin 748, or under control conditions. Relative expression ( RQ ) values were normalized to the control condition for every independent experiment. Bars represent the mean of three independent experiments (± SEM). s16 ribosomal protein mRNA served as endogenous control. b , d , f bEnd5 total VE-cadherin, β-catenin, and ZO-1 protein amounts were determined by Western blot analysis after 48 h in culture. Protein levels were normalized to the endogenous control protein β-actin. Values were normalized to the control condition for every independent experiment. Bars represent the mean of three (VE-cadherin, β-catenin) and six (ZO-1) independent experiments (± SEM). * P < 0.05 ( n.s. not significant)

Article Snippet: The following primary antibodies were used for immunohistochemistry (IHC) and immunofluorescence (IF) staining: monoclonal mouse anti-chicken agrin antibodies 5B1 (1:500) and C3 (1:200; Reist et al. ), polyclonal rabbit anti-mouse agrin serum 204 (1:1000; Eusebio et al. ), polyclonal rabbit anti-claudin-5 (1:100; 34–1,600, Zymed), polyclonal rabbit anti-ZO-1 (1:100; 61–7,300, Invitrogen), polyclonal rabbit anti-ZO-1-associated Y-box factor (ZONAB, Mid; 1:150; 40–2,800, Invitrogen), polyclonal rabbit anti-ZO-2 (1:100; 71–1,400, Zymed), polyclonal rabbit anti-occludin (1:50; 71–1,500, Zymed), polyclonal rabbit anti-β-catenin (1:2000; C2206, Sigma), monoclonal rat anti-VE-cadherin (11D4.1, hybridoma supernatant; Gotsch et al. ; kindly provided by Prof. Dietmar Vestweber, Münster, Germany), monoclonal rat anti-JAM-A (BV12; Bazzoni et al. ; kindly provided by Prof. Elisabetta Dejana, Milan, Italy), polyclonal rabbit anti-laminin (1:1000 for IHC, 1:4000 for IF; Z0097, DakoCytomation), polyclonal rabbit anti-caveolin (1:450; 610,059, BD Transduction Laboratories), biotinylated polyclonal goat anti-mouse IgG (1:200; BA9200, Vector).

Techniques: Expressing, Polymerase Chain Reaction, Cell Culture, Western Blot

Journal: Cell and Tissue Research

Article Title: The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

doi: 10.1007/s00441-014-1969-7

Figure Lengend Snippet: Agrin −/− pMBMECs show reduced junctional localization of VE-cadherin, β-catenin, and ZO-1. a , b Primary mouse brain microvascular endothelial cells (pMBMECs) were isolated from rescued agrin knock-out (c-mag B8 //agrn −/− ) or control littermates (c-mag B8 //agrn +/+ ), and after 7 days in culture, immunofluorescence staining for agrin was performed. The presence of the extracellular matrix protein agrin is visible by specific binding of the fluorochrome-labeled anti-agrin antibody in control cells ( white signal in b ), and the absence of this binding on agrin-knock-out endothelial cells (no fluorescence signal in a ). Micrographs from one representative experiment out of five are shown. Bar 50 μm. c Permeability of agrin-deficient (c-mag B8 //agrn −/− ) and control pMBMECs (c-mag B8 //agrn +/+ ) was measured after 7 days in culture for 3-kDa Dextran. Bars represent the mean of seven independent experiments (± SEM). d–i After 7 days, pMBMECs isolated from rescued agrin knock-out (c-mag B8 //agrn −/− ) or control littermates (c-mag B8 //agrn +/+ ) were stained for VE-cadherin ( d , e ), β-catenin ( g , h ), and ZO-1 ( j , k ), and the junctional IF signal quantified by measuring the gray values at the junctions ( f , i , l ). Micrographs of one representative experiment out of five are shown. Bars 50 μm. The gray values of the knock-out cells were normalized to value of the control cells, which was set to 1. Each symbol represents the mean gray value of all five independent experiments, and the horizontal lines indicate the means of all experiments performed. * P < 0.05

Article Snippet: The following primary antibodies were used for immunohistochemistry (IHC) and immunofluorescence (IF) staining: monoclonal mouse anti-chicken agrin antibodies 5B1 (1:500) and C3 (1:200; Reist et al. ), polyclonal rabbit anti-mouse agrin serum 204 (1:1000; Eusebio et al. ), polyclonal rabbit anti-claudin-5 (1:100; 34–1,600, Zymed), polyclonal rabbit anti-ZO-1 (1:100; 61–7,300, Invitrogen), polyclonal rabbit anti-ZO-1-associated Y-box factor (ZONAB, Mid; 1:150; 40–2,800, Invitrogen), polyclonal rabbit anti-ZO-2 (1:100; 71–1,400, Zymed), polyclonal rabbit anti-occludin (1:50; 71–1,500, Zymed), polyclonal rabbit anti-β-catenin (1:2000; C2206, Sigma), monoclonal rat anti-VE-cadherin (11D4.1, hybridoma supernatant; Gotsch et al. ; kindly provided by Prof. Dietmar Vestweber, Münster, Germany), monoclonal rat anti-JAM-A (BV12; Bazzoni et al. ; kindly provided by Prof. Elisabetta Dejana, Milan, Italy), polyclonal rabbit anti-laminin (1:1000 for IHC, 1:4000 for IF; Z0097, DakoCytomation), polyclonal rabbit anti-caveolin (1:450; 610,059, BD Transduction Laboratories), biotinylated polyclonal goat anti-mouse IgG (1:200; BA9200, Vector).

Techniques: Isolation, Knock-Out, Immunofluorescence, Staining, Binding Assay, Labeling, Fluorescence, Permeability