antigen 2  (R&D Systems)

Journal: bioRxiv

Article Title: Pericytes require physiological oxygen tension to maintain phenotypic fidelity

doi: 10.1101/2024.08.28.606682

Figure Lengend Snippet: Pericytes obtained from mouse lung, brain, bone, and liver were seeded at passage 1 for immunofluorescence study. A panel of pericyte identification markers including NG2, MYH11, PDGFRb, and ACTA2 were used to characterize pericyte marker expression in different organs. One representative image is displayed from a total of 3 independent pericyte batches. Scale Bar = 50 μm.

Article Snippet: The following primary antibodies were used; rabbit anti-MYH11 (ab224804, Abcam), rabbit anti-PDGFRb (ab32570, Abcam), recombinant Alexa Fluor 647 anti-NG2 (ab283639, Abcam), and mouse anti-ACTA2 FITC conjugated (F3777, Millipore Sigma).

Techniques: Immunofluorescence, Marker, Expressing

Journal: Frontiers in endocrinology

Article Title: Functional Characterization of the Human Islet Microvasculature Using Living Pancreas Slices.

doi: 10.3389/fendo.2020.602519

Figure Lengend Snippet: FIGURE 1 | Pericytes cover human and mouse islet capillaries and express aSMA. (A) Maximal projection of confocal images of a human (left panel; 16-year-old individual) and a mouse islet (right panel; 2 months old) in pancreatic sections immunostained for pericytes (neuron-glial antigen 2 (NG2), green) and endothelial cells (CD31, red). Pericytes cover mouse and human islet capillaries. Their densities in islets are higher than in surrounding exocrine tissues. (B) Maximal projection of confocal images of a human islet immunostained for insulin (red) and smooth muscle actin alpha isoform (aSMA, green). The donor was a 16-year-old individual. Scale bar = 50 mm. (C) Maximal projections of confocal images of human (left panel; 16 year old) and a mouse islet (right panel; 2 months old) in pancreatic sections immunostained for pericytes (NG2, green) and aSMA (red). (D) Maximal projections of confocal images of a region within a human islet immunostained for NG2 (green), aSMA (red) and insulin (blue). Human donor was a 44-year-old individual. (E) Quantification of the islet aSMA density, which is the % of aSMA immunostained area divided the total islet area (N = 24 human islets from 7 non-diabetic human donors (male and female; ages: 15–55 years old); N = 37 mouse islets from 6 C57BL6 mice; 2–18 months old). *p < 0.05 (unpaired t-test). (F, G) Mander’s (M1 and M2) coefficients reflecting the colocalization between NG2 and aSMA immunostainings. (F) is the fraction of aSMA-positive immunostaining that colocalizes with NG2 and (G) is the fraction of NG2 -positive immunostaining that colocalizes with aSMA. Panel G has been partially published in (11). We analyzed 21 human islets from 7 non-diabetic human donors (male and female; ages: 15-55 years old) and 31 mouse islets from 6 C57BL6 male mice (2–18 months old). (H, I) Zoomed images of NG2 (green) and aSMA (red) labeled cells in human (H) and mouse islets (I) Different types of pericytes can be found in human islets. We named “SMC” pericytes those cells with more circumferential cytoplasmic processes found on the surface of human islet feeding arterioles (left and middle panels, H) to distinguish them from pericytes that were present in the islet parenchyma (islet pericytes, right panels in H and I). “SMC” pericytes and some of human and mouse islet pericytes express aSMA. Scale bars = 50 mm (A–C) and 10 mm (H, I).

Article Snippet: To identify pericytes in situ, slices were incubated for 2 h with a fluorescent-conjugated antibody against NG2 (final dilution 1:50, R&D Systems, cat. nr. Fab2585R).

Techniques: Immunostaining, Labeling

Journal: Frontiers in endocrinology

Article Title: Functional Characterization of the Human Islet Microvasculature Using Living Pancreas Slices.

doi: 10.3389/fendo.2020.602519

Figure Lengend Snippet: FIGURE 5 | Vasoactive substances change [Ca2+]i levels in human and mouse islet mural cells. (A) Confocal images of a human islet in a living pancreas slice showing fluorescent NG2 antibody (NG2-alexa647; magenta) labeled pericytes in situ and fluo-4 loaded cells (green) in living slices. NG2 is a proteoglycan expressed at the plasma membrane of pericytes. Slices Scale bars = 20 mm. (B) Maximal projection of confocal images of a human islet in a pancreatic section immunostained for insulin (blue) and pericytes using two different anti-NG2 antibodies: a non-conjugated one (red; used in Figure 1) and another conjugated to Alexa647 (green). NG2-alexa647 recognizes pericytes. (C) Confocal images of regions within the human islet shown in (A). Different islet mural cells incorporate the calcium indicator Fluo4 (green) and can be visualized with NG2-alexa647 antibody (magenta), such as “SMC” pericytes (upper panel) and islet pericytes (lower panel). (D) Changes in Fluo4 fluorescence of cells shown in (C) reflecting changes in [Ca2+]i in “SMC” pericytes (upper panels) and islet pericytes (lower panels) under basal conditions (3 mM glucose concentration, 3G, left panels), after 3 min with NE (middle panels) and 5 min with ET-1 (right panels). Pseudocolor (LUT fire) images are shown to better illustrate absolute changes in fluorescence. Arrows indicate different cells responding to the stimuli, and the * shows the vessel lumen. (E) Traces showing relative changes in fluorescence induced by norepinephrine (NE) in individual islet mural cells: “SMC” pericytes (black traces), pericytes at the islet border (dark gray traces) and pericytes in the islet parenchyma (light gray traces). (F) Quantification of changes in fluorescence induced by NE, ET-1 or adenosine (ADO) for individual mural cells in human and mouse islets: “SMC” pericytes in human islets (dark gray box-plot), human islet pericytes (light gray box-plot) and mouse islet pericytes (white box-plot). Maximum (peak) amplitude values were taken before (-) and at the end of stimulus application (+). [Ca2+]i data of mouse islet pericytes was from experiments using mice that expressed the genetically encoded calcium indicator GCaMP3 in pericytes (11). The numbers of cells analyzed are shown above the box-plots. Data are from three different non-diabetic individuals, three different mice, at least one islet per individual. *p < 0.05 (paired t-test, comparisons with corresponding values before stimulus application).

Article Snippet: To identify pericytes in situ, slices were incubated for 2 h with a fluorescent-conjugated antibody against NG2 (final dilution 1:50, R&D Systems, cat. nr. Fab2585R).

Techniques: Labeling, In Situ, Clinical Proteomics, Membrane, Concentration Assay