Two weeks postinjury, rats were sacrificed by transcardial perfusion with 0.9% saline followed by 4% formaldehyde in 0.1 M PB (pH 7.0), under deep ketamine cocktail anesthesia (100 mg/kg ketamine, 2.2 mg/kg acepromazine, and 0.48 mg/kg atropine, diluted in 0.9% sterile saline). Three animals were assessed for expression of neuronal and sensory markers; data are reported as proportion of RFP NSCs expressing each marker (Table 3). The cord was segmentally cut at 1-cm increments: lesion site (L, centered at T10 transection site), 1 and 2 cm rostral, and 1 cm caudal to L, embedded together in gelatin and sectioned at 40 μm on a vibratome. Every sixth section was used for immunostaining. Primary antibodies for immunostaining are detailed in Table 1 and secondary antibodies are discussed above. In addition to use of preimmune serum controls for immunostaining, sections of heart, lung, liver, kidney, and muscle tissue were simultaneously stained in the same vial with the spinal cord sections.
