ra3 6b2 neutrophils s100a8 a100a9 goat rabbit r d systems abcam af3059 ab105472 macrophages iba1 f4 80 rabbit rat wako biolegend 019  (R&D Systems)

Journal: eLife

Article Title: Injury-induced perivascular niche supports alternative differentiation of adult rodent CNS progenitor cells

doi: 10.7554/eLife.30325

Figure Lengend Snippet: ( A ) Density of OPCs positive for active caspase three in VN is similar to their density in non-VN during early remyelination (n = 3, mean ± SEM). ( B ) Identification of genetically labelled PDGFRA+ OPCs with distinct phenotype of YFP+/Sox10+/Olig2- in vascular (astrocyte-free) areas of the EB-induced lesions in spinal cords of Pdgfra-CreERT2 mice (arrowheads, scale bar 50 μm). Cre recombination was induced by administering tamoxifen on each of four consecutive days. Four days after tamoxifen induction, focal demyelination was induced in spinal cord white matter by injection of a demyelinating toxin. Immunohistochemistry was performed on sections at 14 days post-lesion. ( C ) Remyelination is temporally accompanied by blood vessel reconstruction and regeneration of the BBB. Blood vessels were marked with von Willebrand factor (vWF) or CD31-positive endothelial cells and their basement membrane by RCA120 binding (representative images, scale bar 100 μm). Detection of SMI71, a protein produced by endothelial cells only under intact blood-brain or blood-nerve barrier conditions allowed us for identification of reconstructed brain blood barrier (BBB). ( D ) Astrocytes gradually repopulate lesion areas. We found that at six dpl astrocytes did not exist in the core of lesion since they were likely killed by ethidium bromide. However, at 10 dpl we detected astrocytes migrating from the rim to core of the lesion, at 14 dpl they were present in the vicinity of large blood vessels whereas by 28 dpl repopulate entire remyelinated area. Nevertheless, at every time points reactive gliosis was significantly higher at the edges of lesion. ( E ) The response and infiltration of Iba1+ inflammatory cells appears equal in regards of cell morphology, number and distribution in both, vascular and non-vascular niche at early remyelination (RCA120 marks blood vessels, representative images, scale bar 50 μm). ( F ). Perivascular SCs form a network with no discontinuity of basement membrane of the outer wall of blood vessel re-expressing SMI71, 28 dpl (representative image, scale bar 20 μm).

Article Snippet: Primary antibody used: Caspase3 R and D Systems AF835-SP; CTGF Abcam ab6992; GFAP DakoCytomation Z0334; GFAP BD Biosciences 556330; GFP Abcam ab6673; MPZ Abcam ab39375; Neurofilament NF DakoCytomation M0762; Olig2 Abcam ab33427; Olig2 Millipore MABN50; Periaxin Gift from Prof. Peter Brophy, University of Edinburgh, UK; SMI71 Calbiochem NE1026; Sostdc1 Abcam 99340; Sox10 Gift from Dr. Michael Wegner, Universität Erlangen-Nürnberg, Erlangen, Germany; Sox2 Santa Cruz Biotechnology sc-17320; vWF Abcam ab6994; Iba1 Wako 019–19741, MBP R@D Systems MAB42282, tRFP Evrogen AB233.

Techniques: Injection, Immunohistochemistry, Membrane, Binding Assay, Produced, Expressing