polyclonal rabbit anti-mouse-pecam-1 antibody  (Santa Cruz Biotechnology)

Journal: bioRxiv

Article Title: Amyloid β induces cardiac dysfunction and neuro-signaling impairment in the heart of an Alzheimer’s disease model

doi: 10.1101/2023.07.11.548558

Figure Lengend Snippet: Top panels: Representative digital images (scale bar 50μm) showing endothelial cells (stained with CD31, in orange) and Aβ (in green). Bottom panels: representative digital images (scale bar 50μm) showing smooth muscle cells by α-Smooth muscle alpha-actin staining (αSMA, in red) and Aβ (in green) in cardiac sections from WT and Tg2576 mice. n=3-4 mice/group. WT mice are represented in the left panels and Tg2576 mice in the right panels.

Article Snippet: Via indirect immunofluorescence procedure, sections were analyzed using rabbit polyclonal antibodies against CD31 (AF3628-R&D Systems, 1:30) and α-smooth muscle actin (α-SMA, ab32575-Abcam, 1:500) at 4°C overnight, followed by specific secondary antibodies conjugated with cyanine 3 or cyanin 5 respectively, to stain vascular network.

Techniques: Staining

Journal: bioRxiv

Article Title: Systemic inflammation differentially modulates the levels of EBI2 and CH25H/CYP7B1 enzymes in the brain microvascular cells

doi: 10.1101/2023.04.16.537063

Figure Lengend Snippet: A . Schematic representation of the synthetic and degrading pathway of 7α,25OHC, the endogenous ligand for EBI2. B . EBI2 was detected in the isolated brain microvessels. The highest co-colocalisation was detected with the pericyte (PDGFRβ+ 0.99 +/- 0.003) and EC (CD31 + 0.97 +/-0.09) markers. C. The first enzyme in the 7α,25OHC synthetic pathway, CH25H, was detected in CD31 + (ECs), GFAP+ (astrocytic endfeet) and PDGFRβ+ (pericytes/smooth muscle cells) cells with the highest co-localisation with the EC marker CD31 (0.73 range 0.33) and lowest with the pericyte marker PDGFRβ (0.13 +/- 0.22). D . The second enzyme, CYP7B1, moderately co-localised with all three cell markers, CD31 (0.59 +/-0.83), GFAP (0.45 +/-0.72) and PDGFRβ (0.46 +/-0.47). E . The 7α,25OHC degrading enzyme, HSD3B7 mostly co-localised with the EC+ (0.87 +/-0.16) and pericyte/smooth muscle cells (0.81 +/-0.52) cells and least with astrocytes (0.16 +/-0.52). Scale 50 μm. Data shown as violin plot with median (solid line) and quartiles (dotted line); 1=complete co-localisation (solid line) and 0 =no co-localisation, N =microvessels isolated from 8 mouse brains.

Article Snippet: Primary antibodies used for mouse microvessels (1:100) were: goat polyclonal EBI2 (RRID: AB_10903697, ab121001, Abcam), rabbit polyclonal CH25H (600-401-MM8, ThermoFisher), mouse monoclonal CYP7B1 (OTI1G7) (TA807549, ThermoFisher), rabbit polyclonal HSD3B7 (RRID: AB_10856786, BS-2366R, ThermoFisher), rabbit monoclonal GFAP (RRID: AB_2631098, 12389, CellSignalling), mouse monoclonal GFAP antibody (1:200) (RRID: AB_2827276, SAB5201104, Sigma-Aldrich), mouse monoclonal CD31 (PECAM-1) (RRID: AB_10596359, BMS137, eBioscience), rabbit polyclonal CD31 (PECAM-1) (RRID: AB_10981955, PA5-16301, ThermoFisher), goat polyclonal PDGFRβ (RRID: AB_2162633, AF1042, R&D Systems), rabbit monoclonal PDGFRβ (RRID: AB_10985851, MA5-15143, ThermoFisher).

Techniques: Isolation, Marker

Journal: bioRxiv

Article Title: Systemic inflammation differentially modulates the levels of EBI2 and CH25H/CYP7B1 enzymes in the brain microvascular cells

doi: 10.1101/2023.04.16.537063

Figure Lengend Snippet: A . The density of EBI2 in CD31 + cells did not increased after LPS treatment. The biggest increase in EBI2 was observed in GFAP+ cells (75% +/-28%) and a moderate increase in PDGFRβ+ cells (34% +/-12%). B . The density of the first enzyme in the 7α,25OHC synthetic pathway, CH25H, increased after LPS by 46% (+/-10) in PDGFR + cells and 38% (+/-7.4%) in GFAP+ cells. The density in CD3 l + (ECs) remained most stable with an increase of 11% (+/-1%) after LPs challenge. C . In contrast, the density of the second synthesising enzyme, CYP7B1, increased mostly in CD31+ cells (72% +/-25%), followed by GFAP+ cells (41% +/-23%) and no change in PDGFRβ+ cells. D . The 7α,25OHC degrading enzyme, HSD3B7, was the least affected by systemic inflammation with density increasing in CD31+ cells by 3.8% (+/-0.56%), 7% (+/-2.4%) in PDGFR + cells and 6.2% (+/-1.2%) in GFAP+ cells. Data presented as mean +/- SEM (N=6 mice) after normalisation to vehicle (=1). Immunostaining of blood vessels in the cortex was performed on brain sections cut in the coronal plane (sections corresponding to levels 70-87 in Allen Mouse Brain Atlas).

Article Snippet: Primary antibodies used for mouse microvessels (1:100) were: goat polyclonal EBI2 (RRID: AB_10903697, ab121001, Abcam), rabbit polyclonal CH25H (600-401-MM8, ThermoFisher), mouse monoclonal CYP7B1 (OTI1G7) (TA807549, ThermoFisher), rabbit polyclonal HSD3B7 (RRID: AB_10856786, BS-2366R, ThermoFisher), rabbit monoclonal GFAP (RRID: AB_2631098, 12389, CellSignalling), mouse monoclonal GFAP antibody (1:200) (RRID: AB_2827276, SAB5201104, Sigma-Aldrich), mouse monoclonal CD31 (PECAM-1) (RRID: AB_10596359, BMS137, eBioscience), rabbit polyclonal CD31 (PECAM-1) (RRID: AB_10981955, PA5-16301, ThermoFisher), goat polyclonal PDGFRβ (RRID: AB_2162633, AF1042, R&D Systems), rabbit monoclonal PDGFRβ (RRID: AB_10985851, MA5-15143, ThermoFisher).

Techniques: Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice

doi: 10.3390/ijms23158693

Figure Lengend Snippet: Vascular development in normoxic and hypoxic developing mouse brains with and without rhGH and rhEPO treatment. The vessel area in the parietal cortex ( A , B ) and hippocampus ( C , D ) was quantified by Pecam-1 IHC after a regeneration period of 48 h ( A , C ) and 7 d ( B , D ). Data are presented as mean ± SEM. *, p < 0.05.

Article Snippet: After heat-induced epitope retrieval, washing with Tris-buffered saline (TBS) containing 0.05% ( v / v ) Tween 20, and blocking with 10% normal goat serum, sections were incubated overnight at 4 °C with 1.0 µg/mL mouse monoclonal anti-mouse-OCLN (Santa Cruz Biotechnology; Heidelberg, Germany) and 0.5 µg/mL polyclonal rabbit anti-mouse-PECAM-1 antibody (Santa Cruz; Heidelberg, Germany) diluted in Dako Antibody Diluent (Agilent; Waldbronn, Germany) containing 10% normal goat serum.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice

doi: 10.3390/ijms23158693

Figure Lengend Snippet: Quantification of OCLN + area by immunofluorescence analysis of OCLN and PECAM-1 protein in normoxic and hypoxic developing mouse brains with and without rhGH and rhEPO treatment after a regeneration period of 48 h ( A , C ) and 7 d ( B , D ) in the parietal cortex ( A , B ) and hippocampus ( C , D ). Data are presented as mean ± SEM. *, p < 0.05.

Article Snippet: After heat-induced epitope retrieval, washing with Tris-buffered saline (TBS) containing 0.05% ( v / v ) Tween 20, and blocking with 10% normal goat serum, sections were incubated overnight at 4 °C with 1.0 µg/mL mouse monoclonal anti-mouse-OCLN (Santa Cruz Biotechnology; Heidelberg, Germany) and 0.5 µg/mL polyclonal rabbit anti-mouse-PECAM-1 antibody (Santa Cruz; Heidelberg, Germany) diluted in Dako Antibody Diluent (Agilent; Waldbronn, Germany) containing 10% normal goat serum.

Techniques: Immunofluorescence

Journal: International Journal of Molecular Sciences

Article Title: Further Evidence of Neuroprotective Effects of Recombinant Human Erythropoietin and Growth Hormone in Hypoxic Brain Injury in Neonatal Mice

doi: 10.3390/ijms23158693

Figure Lengend Snippet: Representative photomicrographs of PECAM-1 (green) and OCLN (red) protein co-staining of vascular endothelial cells in hypoxic developing mouse brains ( B , D , F , H ) compared to normoxic controls ( A , C , E , F ) after a 48 h regeneration period in NT (A,B), VT ( C , D ), rhGH- ( E , F ), and rhEPO-treated brains ( G , H ) in the parietal cortex. Blue, 4′,6-diamidino-2-phenylindole (DAPI) nuclear counterstain.

Article Snippet: After heat-induced epitope retrieval, washing with Tris-buffered saline (TBS) containing 0.05% ( v / v ) Tween 20, and blocking with 10% normal goat serum, sections were incubated overnight at 4 °C with 1.0 µg/mL mouse monoclonal anti-mouse-OCLN (Santa Cruz Biotechnology; Heidelberg, Germany) and 0.5 µg/mL polyclonal rabbit anti-mouse-PECAM-1 antibody (Santa Cruz; Heidelberg, Germany) diluted in Dako Antibody Diluent (Agilent; Waldbronn, Germany) containing 10% normal goat serum.

Techniques: Staining