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. Author manuscript; available in PMC: 2010 Mar 15.
Published in final edited form as: J Neurosci Methods. 2008 Nov 27;177(2):440–447. doi: 10.1016/j.jneumeth.2008.11.013

Figure 3. Fluoro-Jade C staining reveals trends in tissue health.

Figure 3

Fluoro-Jade C staining labels dead and degenerating neurons regardless of the type of degeneration process (Schmued et al., 2005). (A-F) Montage images of VNO (A-C) and AOB (D-F) sections taken from dissection-only (A, C), physiology chamber plus microsuction (B, E), and unoxygenated, warmed aCSF (C, F, “unperfused”) conditions are shown for reference. (A, D) In control sections, very few bright, punctate areas were present in the vomeronasal epithelium (A) or the AOB glomerular, mitral, or granule cell layers (D). (B, E) Following 6 (VNO, panel B) or 4 (AOB, panel E) hours in the physiology chamber at 33 – 35 °C, relatively few punctate areas were visible in the glomerular and mitral cell layers. (C, F) When VNO-AOB ex vivo preparations were placed in unperfused, unoxygenated aCSF at 33 – 35 °C, we saw substantial global increases in Fluoro-Jade C staining, indicative of widespread neuronal death and degeneration. (G) Quantification of punctate staining in the vomeronasal epithelium after 6 hours showed no increase in cell degeneration in the perfusion chamber compared to dissection-only controls. (H) Quantification of punctate staining in the AOB glomerular and mitral cell layers indicated the perfusion chamber provided significant protection from damage (blue symbols) compared with the unoxygenated tissue (green symbols), especially at the 4 and 6 hour time points (p < 0.001, n = 3). (I) We encountered evidence of degeneration in the granule cell layer under normal superfusion conditions (blue symbols). To encourage perfusion through deeper tissue layers, we drew solution through the tissue using a microsuction device (E). Microsuction for 4 hours in the tissue chamber reduced mean staining in the granule cell layer to a level intermediate between “unperfused” (p = 0.2, n = 5) and “dissection only” (p = 0.5, n = 5; (H, I, red symbols). Abbreviations: VE, vomeronasal epithelium; L, vomeronasal lumen; GL, glomerular layer; MCL, mitral cell layer; GCL, granule cell layer; LOT, lateral olfactory tract.