sheep anti cd54  (R&D Systems)

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Venular and non-venular endothelial cells in adult human skin can be distinguished by conventional markers. ( A ) Expression patterns of the venular marker ACKR1, the adhesion molecules ICAM1 and SELP and the arterial marker HEY1. ( B ) Representative section (upper) and whole-mount staining (lower) of the pan-endothelial marker ERG (green), HEY1 (red) and SELP (white) distinguish individual BV compartments. White arrowheads, empty arrowheads and asterisks indicate the SELP+HEY1−, SELP−HEY1+ and SELP+HEY1+ populations, respectively. Scale bars: 100 µm.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Expressing, Marker, Staining

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Gene signatures peculiar to the venular compartment. ( A ) EFNB2 expression measured by scRNA-seq (left) and immunofluorescence staining (right) denoting its abundance across different BEC subpopulations and its exclusive expression in human dermal BVs (white arrowheads). ( B ) Representative confocal images of skin sections (upper) and whole-mount skin blocks (lower) stained for ERG (green), EFNB2 (red) and ICAM1 (white). The unique composition of EFNB2 and ICAM1 expression enables the discrimination of arterioles (empty arrowheads), post-capillary venules (*) and venules (white arrowheads). ( C ) EGR2 expression is most enriched in the post-capillary venule cluster and is restricted to BVs (white arrowheads). ( D ) Representative section (upper) and whole-mount staining (lower) of ERG (green), EGR2 (red) and ICAM1 (white), highlighting EGR2+ICAM1+ BVs. Scale bars: 100 µm.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Pre-venular capillaries are pinpointed by novel markers. Specific localization of the PVC markers SOX17 ( A ) and PLAUR ( B ) in human dermal BVs (white arrowheads). ( C ) Intersection of ICAM1 (white) and SOX17 (red) highlights the segments of pre-venular capillaries. ( D ) Co-staining of ICAM1 (red) and PLAUR (white) in skin sections (upper) and whole-mount skin blocks (lower) denotes the pre-venular capillary cluster. Scale bars: 100 µm.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Staining

Journal: Cells

Article Title: Novel Blood Vascular Endothelial Subtype-Specific Markers in Human Skin Unearthed by Single-Cell Transcriptomic Profiling

doi: 10.3390/cells11071111

Figure Lengend Snippet: Summary of marker expression in different BEC subtypes.

Article Snippet: After fixation in 4% paraformaldehyde (PFA), skin sections were incubated in blocking solution (5% donkey serum, 0.2% BSA, 0.3% Triton X-100 and 0.05% NaN 3 in PBS) for 1 h. Subsequently, the sections were incubated at 4 °C overnight with the respective primary antibodies, which include rabbit anti-human LYVE-1 (1:100, 102PA50AG, ReliaTech, Wolfenbüttel, Germany), biotinylated goat anti-human LYVE-1 (1:100, BAF2089, R&D, Minneapolis, MN, USA), mouse anti-human CD31 (1:100, clone JC70A, M0823, Dako, Santa Clara, CA, USA), rabbit anti-human CD31 (1:40, GTX110602, GeneTex, Irvine, CA, USA), rabbit anti-human ERG (1:100, ab92513, Abcam, Cambridge, UK), goat anti-human ERG (1:50, NB100-2472, Novus, Centennial, CO, USA), goat anti-human ACKR1 (1:200, NB100-2421, Novus), mouse anti-human ICAM1 (1:100, clone W-CAM-1, MA5-13021, Invitrogen, Waltham, MA, USA), sheep anti-human ICAM1 (1:100, AF720, R&D), rabbit anti-human HEY1 (1:100, ab22614, Abcam), mouse anti-human SELP (1:300, clone AK-6, ab6632, Abcam), goat anti-human EFNB2 (1:100, AF496, R&D), rabbit anti-human EGR2 (1:200, ab43020, Abcam), rabbit anti-human LRG1 (1:200, HPA001888, Atlas Antibodies, Bromma, Sweden), mouse anti-human GJA5 (1:250, clone 2F9A11, 37-8900, Invitrogen), mouse anti-human ASS1 (1:100, clone 2B10, ab124465, Abcam), rabbit anti-human S100A4 (1:200, ab41532, Abcam), goat anti-human SOX17 (1:10, AF1924, R&D) and mouse anti-human PLAUR (1:100, clone 62022, MAB807, R&D).

Techniques: Marker, Expressing