A: Mixed cultures of neonatal rat neurons and glia stained with Mouse monoclonal antibody to Nestin [4D11] M-1385-100 (red), Chicken polyclonal antibody to vimentin C-1409-50 (green) and DNA (DAPI stain, blue). Astrocytes and neuronal stem cells stain strongly and specifically in a clearly filamentous fashion with the Nestin antibody. The filamentous staining pattern is as expected as both Nestin and Vimentin are components of 10nm filaments. Note that some cells contain Nestin, but do not stain strongly for Vimentin and so appear red. Others contain Vimentin and not Nestin and so appear green- these are likely to be fibroblastic or endothelial cells. Some cells express both proteins and so appear yellowish. The presence of Nestin indicates that the cells are developing astrocytes, neuroblasts or undifferentiated neural stem cells. B: Western blot in of developing rat brain (P18) homogenate probed with mouse monoclonal antibody to Nestin M-1385-100. A single strong band running at ~240 kDa is seen. C: Analysis of nesting expression in human neuroblastoma SH-SY5Y by Flow Cytometry. Fixing and Permeabilization of cells: Absolute methanol (10 minutes in ice) and 0.1% Tween-20 in PBS, Blocking: 1% BSA, Primary antibody: Mouse Monoclonal antibody to Nestin (cat # M-1385-100, 2μg per ~10^6 cells) for 30 minutes at room temperature, Secondary antibody: Goat anti-mouse PE labeled secondary antibody (1:100 fold dilution) with incubation for 20 minutes in dark at room temperature. Non-specific Control IgG, clone X63 (cat # M-1249-200) was used as negative control under same conditions (black dashed). Flow cytometry data and results were generated using Orflo MoxiflowTM instrument and protocols.