WNT-5A expression in mouse GFAP
astrocytes. (A) Immunohistochemistry was performed on adult mouse brain sections using an anti-WNT-5A antibody in combination with anti-glial fibrillary acidic protein (GFAP) and ionized calcium-binding adaptor protein1 (IBA1) antibodies as astrocyte and microglia marker, respectively. Merge presents the overlay of IBA1, GFAP, WNT-5A. Size bar −2 μm. The images represent a maximum intensity projection of a Z-stack of 5 μm thickness. The white square marked ‘B’ indicates the area magnified in B: (B) Close up of a GFAP+ astrocyte reveals the expression of WNT-5A in this cell type. (C) shows immunoblot detection of recombinant WNT-5A (rWNT-5A; 375 ng/lane) in comparison to lysates from mouse primary microglia and mixed astrocyte cultures. β-actin serves as a loading control. (D) The bar graph depicts expression levels of WNT-5A mRNA in mouse primary microglia and mixed astrocyte cultures measured by QPCR. Data are normalized to GAPDH expression and analyzed with a non-parametric Mann–Whitney test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. n = 4 to 8. (E) shows indirect immunocytochemistry of mixed astrocyte cultures employing anti-GFAP as astrocyte and anti-CD11b as microglia markers. DAPI is used as nuclear counterstain. Image represents a maximum image projection of an 8 μm Z-stack. Size bar 20 μm. The frame shows 63 cells in total and 10 CD11b-positive microglia (arrows). Routinely 10% to 18% microglia were observed (n = 4). DAPI, 4',6-diamidino-2-phenylindole; GADPH, glyceraldehyde 3-phosphate dehydrogenase; n, number.