Anti-CD31 antibody (ab28364)

CLARITY-based immunochemistry showing Nerve fibers and vessels in the mesentery.

A) Catecholaminergic nerve fibers stained for TH. (B) Nerves stained for the pan-neuronal marker TUJ-1. (A,B) Illustrate the integration of nerve fibers in the mesentery (asterisk) into the intramural neuronal networks of the gut. (C) Depicts blood vessels stained with the endothelial marker CD31 (using ab28364 at 1:50) and shows the branching of vessels coming from the mesentery (asterisk) and fine capillaries in the villi (confocal image stack 10x/0.3 objective, z-range 1047 μm). (D–F) Show the innervation of blood vessels in the mesentery ((D) CD31 (using ab28364 at 1:50), (E) TUJ-1, (F) Merge). (G–I) Identifies the very fine sympathetic nerve fibers of arterioles by specific catecholaminergic staining. Scale bars: (A–C) 500 μm; (D–F) 100 μm; (G–I) 100 μm.

Displaying the mesentery and intact gut in one preparation is a major improvement in visualizing the neuronal and vascular interface between the alimentary tract and its extrinsic supply, rendering CLARITY-based immunochemistry a promising tool going beyond previous whole mount preparations.

D can be visualized  Supplementary video 6 to fully appreciate the three-dimensional tissue composition.

ab28364 at 1/50 dillution staining CD31 in human tonsil tissue section by Immunohistochemistry (Formalin/ PFA fixed paraffin embedded tissue sections).

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Anti-CD31 antibody (ab28364) at 1/50 dilution staining mouse tissue sections by Immunohistochemistry (Formalin-fixed paraffin-embedded sections). Following heat mediated antigen retrieval the tissue was formaldehyde fixed and blocked with serum. The tissue was incubated with the primary antibody for 20 minutes. A biotinylated goat anti-rabbit antibody was used as the secondary. The endothelium of vessels of any tissues are positive.

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Representative images of cross sections with CD31 staining in red, nucleus in blue. White dotted lines indicate the graft wall. Scale bar = 100 μm.

Explanted grafts from C57/BL6 mice were fixed overnight in paraformaldehyde (2%) at room temperature. Samples were dehydrated through an ethanol gradient and embedded in paraffin and sectioned at 5 μm transversely from proximal anastomosis to distal anastomosis. For immunohistochemistry staining, sections were deparaffinised and stained with antibody against CD31 (Abcam, ab28364, 1:200) for endothelial cells.

ab28364 staining CD31 in mouse xenograft tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with paraformaldehyde, cut into 20 micron slices and blocked with 1% BSA for 60 minutes at 21°C. Antigen retrieval was by heat mediation in a Tris/EDTA pH9 buffer. The sample was incubated with primary antibody (20 ug/mL) at 4°C for 16 hours. A HRP-conjugated goat anti-rabbit polyclonal (2 ug/mL) was used as the secondary antibody.

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GFP positive cells (green) were colocalized with CD31 (red). Nuclear staining with DAPI is blue.

For immunofluorescence staining, the skin samples were fixed in 4% paraformaldehyde and embedded in paraffin. Skin sections were stained with an anti-GFP antibody. The skin sections were also treated with primary Abs against CD31 (Abcam, ab28364, diluted 1:300). Secondary antibodies conjugated to rhodamine-isothiocyanate were used for fluorescence detection on a confocal laser scanning fluorescence microscope (Zeiss, German).

Opn^-/-; OPN (Osteopontin) knock-out mice.