Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: Endothelial-specific eGFP expression in embryoid bodies (EBs) derived from PECAM/eGFP ES cells. ( A – C ) Fluorescence images show eGFP + clusters at day 6 ( A ) and early vascular network formation in EBs at day 8 ( B ) and day 10 ( C ) of suspension culture. Arrow heads indicate eGFP + sprouts. ( D – F ) In plated EBs distinct sprouts can be observed at day 10 + 4 ( D ) resulting in a complex vascular network in the center ( E ) and the periphery ( F ) of the EB at day 10 + 7. ( G – H ) Immunofluorescence stainings of vascular networks reveal the co-localization of native eGFP and PECAM expression. Green = native eGFP, red = PECAM, blue = Hoechst; bars = 200 µm ( A – E ), 100 µm ( F ), 50 µm ( G – I ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: Expressing, Derivative Assay, Fluorescence, Immunofluorescence

Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: EGFP expression in PECAM/eGFP embryos at different developmental stages. ( A ) Fluorescence picture demonstrates bright native eGFP expression in network-like structures of embryos at E9.5. ( B ) PECAM-DAB whole mount staining of littermates shows a good overlap with eGFP + structures in ( A ) confirming PECAM-specific eGFP expression. ( C ) In cryosections of E9.5 embryos co-localization of native eGFP (green) and PECAM staining (red) was observed. Strong eGFP (green) and PECAM (red) expression were identified in the branchial arch (BA), branchial arch artery (BAA), outflow tract of the heart (OFT), common atrial chamber (CAC), common ventricular chamber (CVC) as well as in intersomitic vessels (ISV). The dotted white line defines the surface of the embryo. ( D – H ) Sections of embryos at E15.5 reveal a strong eGFP expression in the developing vasculature ( D ), in particular in the heart ( E ), the lung (F ), the gut ( G ), and the paw ( H ). ( I – K ) Immunofluorescence stainings of heart sections derived from a E15.5 embryo confirms PECAM-specific eGFP expression in blood vessels. Green = native eGFP, brown = PECAM-DAB, red = PECAM ( C , J , K ), autofluorescence (AF) ( D – H ), blue = Hoechst; bars = 500 µm ( A – C ), 100 µm ( D ), 200 µm ( E – H ), 50 µm ( I – K ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: Expressing, Fluorescence, Staining, Immunofluorescence, Derivative Assay

Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: EGFP expression in organs of PECAM/eGFP adult mice. ( A–L) Macroscopic images of organs from adult PECAM/eGFP mice illustrate strong native eGFP expression in network-like structures in the brain ( A ), the white rectangle in ( A ) defines the basilar artery shown in a close up in ( B ). In the eye, limbal vessels in the transition zone between cornea and sclera ( C ) as well as retinal vessels ( D ) exhibit a strong eGFP signal. EGFP expression was also found in the skin ( E ), the skeletal muscle ( F ), heart ( G ), lung ( H ), liver ( I ), kidney ( J ), gut ( K ) and uterus ( L ). Green = native eGFP; bars = 1000 µm ( A ), 100 µm ( B , E – L ), 200 µm ( C , D ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: Expressing

Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: PECAM-specific eGFP expression in different organs of PECAM/eGFP adult mice. ( A – L ) Immunofluorescence stainings with an anti-PECAM antibody reveal prominent PECAM-specific native eGFP expression in the endothelium of the vasculature in the skin ( A – C ), in the lung (note that airway epithelium is eGFP − , ( D – F ), in the kidney (glomeruli and straight vessels, ( G – I ) and the heart ( J–L ). Co-localization of native eGFP fluorescence and PECAM staining was also found in endothelial cells of larger coronary arteries (inset, L ). ( M ) Statistical analysis of eGFP and PECAM signals in myocardial sections shows co-localization in cardiac endothelial cells. ( N ) Flow cytometry analysis of PECAM-PE + cells after MACS reveals successful purification of endothelial cells from adult hearts (before: before sorting, −: negative fraction, + : positive fraction). ( O ) Western blot analysis of these isolated PECAM + cells confirms eGFP expression in PECAM/eGFP mice but not in WT mice (the membrane was cut at about 37 kDa to enable incubation with the two different antibodies against β-actin and eGFP), **p < 0.01, ***p < 0.001 (One way ANOVA, Tukey’s post hoc test, (N). Green = native eGFP, red = PECAM, blue = Hoechst; bars = 50 µm ( A–F , J–L ), 25 µm ( G – I ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: Expressing, Immunofluorescence, Fluorescence, Staining, Flow Cytometry, Purification, Western Blot, Isolation, Incubation

Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: EGFP expression in lymphatic tissue and bone marrow. ( A – D ) Sections of PECAM/eGFP lymph node. Native eGFP expression ( A ) shows a good overlap with PECAM staining ( B ), whereas lymphatic vessels stained with LYVE1 ( C ) are eGFP − . ( E – P ) Immunofluorescence stainings of bone marrow tissue. ( E – L ) Native eGFP is strongly co-localized with PECAM + EMCN − arteries (white arrows) and PECAM + EMCN + sinusoids (white arrowheads). ( I – P ) Roundish eGFP + cells can be identified as blood cells by their strong PECAM and CD45 signal (yellow arrowheads). ( Q – S ) Statistical analysis of eGFP, PECAM and CD45 expression in arteries, sinusoids and blood cells. Green = native eGFP, red = PECAM ( B , D , F – H , J – L ,), CD45 ( N – P ), purple = LYVE1, white = EMCN, blue = Hoechst; bars = 50 µm ( A–H ), 25 µm ( I–P ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: Expressing, Staining, Immunofluorescence

Journal: Scientific Reports

Article Title: PECAM/eGFP transgenic mice for monitoring of angiogenesis in health and disease

doi: 10.1038/s41598-018-36039-2

Figure Lengend Snippet: Utility of PECAM/eGFP mice for analysis of angiogenesis assays and in vivo injury model. ( A – C ) PECAM/eGFP aortic rings demonstrate eGFP + sprouts in a collagen gel assay at d8 (A, transversal view), ( B , frontal view) and d10 ( C , higher magnification). ( D – I ) Griffonia Simplicifolia Lectin (red, D – F ) and PECAM stainings (red, G–I ) of aortic sprouts confirm the endothelial character of eGFP + elongated structures. ( J ) Fluorescence picture 3d after myocardial cryoinfarction in PECAM/eGFP mice shows native myocardium with high autofluorescence ( J , top) and the infarcted area with low background fluorescence ( J , bottom). ( K – P ) PECAM staining (red) confirms endothelial-specific eGFP expression in the capillaries of the border zone ( K – M ) and the center ( N – P ) of the cryoinfarction. The white rectangles in ( J ) define magnified regions of consecutive cryosections shown in ( K – M ) and ( N – P ). Green = native eGFP, red = autofluorescence (AF) ( A – C , J ), GSL ( E , F ), PECAM ( H,I,L,M,O,P ), blue = Hoechst; bars = 200 µm ( A – C ), 100 µm ( D – F , G – I ), 500 µm ( J ), 50 µm ( K – P ).

Article Snippet: Purity was examined by flow cytometric quantitation of eGFP + /PECAM-PE + (BD Biosciences, Heidelberg, Germany) cell populations using a CyFlow space flow cytometer (Sysmex Partec, Görlitz, Germany).

Techniques: In Vivo, Fluorescence, Staining, Expressing