Figure 2.

Technical aspects of nestin staining. SVZ sections obtained from the same block (a specimen is illustrated in (C)). (A), epifluorescence microscopy image (green, nestin; blue, nuclear staining with Hoechst). (B), bright field microscopy image. The ependymal cell layer appears at the top of the panels. Note the variation of the thickness of the NPCL; it is thicker close to larger subventricular vessels (lines in (A) and (B)). The observation of this relationship is facilitated by the magnification employed here; vessels cut transversely and longitudinally (arrowheads) and the perivascular region can be simultaneously observed. In addition, in the less vascularized areas of the SVZ, the NPCL is significantly narrower (arrow in (A)). We identified a fine anucleated layer beneath the ependymal cell layer (asterisks). One technical aspect is the persistence of the autofluorescence of red blood cells in the vessels (A). However, it is easy to distinguish between the autofluorescence of red blood cells and the nestin staining. Importantly, the results obtained by the immunofluorescent method (A) regarding the anatomical distribution of nestin-positive cells (NPCs) in a large brain area (in this case, the SVZ) match the results obtained by the immunoenzymatic method (B). Similarly, the morphology of NPCs revealed by the immunoenzymatic method (arrows in (D)) match the morphology of NPCs revealed by the immunofluorescent method as analyzed with epifluorescence microscopy (arrows in (E)) or confocal microscopy (arrows in (F)). Despite the non-specific background staining, these methods together highlight the specificity of nestin staining depicted in the following figures. Arrows in (C), superficial blood vessels where the likelihood of the presence of NPCs is higher. Arrowheads in (D) and (E), ependymal layer. Scale bars: (A) and (B) = 200 μm; (D), (E), and (F) = 20 μm.