Journal: PLoS ONE

Article Title: Bone Marrow-Derived Mesenchymal Stem Cells Ameliorate Hepatic Ischemia Reperfusion Injury in a Rat Model

doi: 10.1371/journal.pone.0019195

Figure Lengend Snippet: A . Bright-field image. B–C . CD29 and CD105 surface antigens are positive. D . CD31 surfice antigen is negative, E . CD34 surfice antigen negative.

Article Snippet: The fluorescein (green, 1∶3000)-conjugated secondary antibody (COSMO BIO, Inc., Tokyo) against CD31, CD34, and CD105 was applied for 30 min. Rhodamine (red, 1∶3000)-conjugated secondary antibody (Rockland immunochemicals, Inc., Tokyo) against CD29 was also used.

Techniques:

Journal: bioRxiv

Article Title: A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

doi: 10.1101/2020.07.25.218149

Figure Lengend Snippet: Inhibition of angiogenesis and vascular leakage with a monoclonal antibody against LRG1. ( a ) Co-cultured HUVECs and fibroblasts were treated with 15C4 or controls for 11 days, allowing for the formation of microtubules. Post-treatment, vessels were stained with anti-CD31 and analysed with AngioSys 2.0. A significant reduction in tubule length was observed with LRG1 blockade by 15C4 (100 μg/mL) compared to IgG controls ( ***P < 0.0001, one-way ANOVA). ( b ) Metatarsals from wild type C57B6 mice were treated with doses of 15C4 as indicated with mouse IgG as a control, and immunostained for CD31 to reveal vessels. A significant reduction in total tubule length was observed in 15C4 treated metatarsals when compared to IgG (* P < 0.05, ** P < 0.01, *** P = 0.0023, one-way analysis of variance). ( c ) C57bl/6 mice with laser-induced choroidal neovascularisation were injected with 15C4 or IgG control immediately following laser burns. Fluorescein angiography was done on day 7. CNV leakage is significantly reduced in eyes treated with 2.5 μg 15C4 compared to IgG controls (one-way ANOVA followed by Tukey’s multiple comparison test, * P < 0.05). ( d ) Wild type mouse retinas were lasered and then after 7 days fixed and processed for analysis using a combination of RNAscope (to detect Lrg1 mRNA) and immunocytochemistry (for CD31 and collagen IV). The merged image shows Lrg1 mRNA expression in the endothelial cells within the lesion.

Article Snippet: Cells were washed with PBS and fixed with ice cold 70% ethanol for 30 min at room temperature, then immunostained with mouse anti-human CD31 (Cellworks).

Techniques: Inhibition, Cell Culture, Staining, Injection, Immunocytochemistry, Expressing

Journal: bioRxiv

Article Title: A Humanized Antibody against LRG1 that Inhibits Angiogenesis and Reduces Retinal Vascular Leakage

doi: 10.1101/2020.07.25.218149

Figure Lengend Snippet: Epitope mapping and development of a human LRG1 knock-in mouse. ( a ) Schematic representation of LRG1 showing the eight leucine-rich repeats (LRRs), with the epitope located between LRRs 6 and 7. The sequence of the human epitope is shown aligned against the orthologous sequences in seven other mammalian species, along with the sequence identity. ( b ) Competition ELISA in which Magacizumab is bound to human LRG1, with titration of the peptide epitopes of the species in A. Only the human and primate peptides were able to compete across the concentration range tested. ( c ) Schematic showing the mouse Lrg1 gene locus, the locus following homologous recombination of the human LRG1 targeting construct, and the locus showing loss of the chimaeric exon 1 following conditional deletion with the Cre recombinase. ( d ) Retinal sections from the human LRG1 knock-in mouse, following laser-induced choroidal neovascularisation. The images show LRG1 mRNA visualised with RNAscope, together with DAPI (nuclei), collagen IV and CD31. The zoomed merge image (bottom left) shows expression of human LRG1 in the vasculature within the lesion.

Article Snippet: Cells were washed with PBS and fixed with ice cold 70% ethanol for 30 min at room temperature, then immunostained with mouse anti-human CD31 (Cellworks).

Techniques: Knock-In, Sequencing, Enzyme-linked Immunosorbent Assay, Titration, Concentration Assay, Homologous Recombination, Construct, Expressing