On the 28th day after SCI, bilateral and stereotaxical injections of biotinylated-dextran amine (BDA; MW 10,000, 10%, 1 μl/site; Molecular Probes, Eugene, OR) were made into the intermediate gray matter of the T7-8 cord segments at distances of 3 – 6 mm rostral to the contusion site (for BDA, one injection/site/mm longitudinal distances) according to previously published work (Deng et al., 2013 (link)).
BDA staining was performed according to earlier reports (Fouad et al., 2001 (link)). Slides were dried in an incubator at 38°C for 1 hr and washed twice for 10 min in 50 mM Tris-buffered saline (TBS), pH 7.4, followed by two 45 min washes with TBS containing 0.5% Triton X-100. Afterwards, the slides were incubated overnight with an avidin–biotin–peroxidase complex in TBS with Triton (ABC Elite, Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Subsequently, a DAB reaction was performed using the Vector DAB kit (SK4100, Vector Laboratories). The reaction was monitored and stopped by extensive washing in water.
BDA staining was performed according to earlier reports (Fouad et al., 2001 (link)). Slides were dried in an incubator at 38°C for 1 hr and washed twice for 10 min in 50 mM Tris-buffered saline (TBS), pH 7.4, followed by two 45 min washes with TBS containing 0.5% Triton X-100. Afterwards, the slides were incubated overnight with an avidin–biotin–peroxidase complex in TBS with Triton (ABC Elite, Vector Laboratories, Burlingame, CA) according to the manufacturer’s protocol. Subsequently, a DAB reaction was performed using the Vector DAB kit (SK4100, Vector Laboratories). The reaction was monitored and stopped by extensive washing in water.
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