Two mice with T8 crush injuries received unilateral injections of BDA into the sensorimotor cortex at 10 weeks post-injury and were perfused with 4% paraformaldehyde 2 weeks later. An approximately 8mm segment of the spinal cord containing the lesion site was sectioned in the sagittal plane on a Vibratome® at 50μm. Sections were incubated for 1 h with avidin and biotinylated HRP (Vectastain ABC kit; Vector Laboratories), washed in PBS, and then reacted with DAB in 50mM Tris buffer, pH 7.6, 0.024% hydrogen peroxide, and 0.5% nickel chloride. The sections were examined under a light microscope while still wet. Serial sections with BDA labeled axons caudal to the injury were selected for electron microscopic analysis.
The selected sections were rinsed in 0.1 M cacodylate buffer and postfixed with 1 % osmium tetroxide in 0.1 M cacodylate buffer for 1 hour, rinsed in ddH20 for 2 × 10 min., dehydrated in increasing serial dilutions of ethanol (70%, 85%, 95%, 100% × 2) for 10 min each, put in propylene oxide (intermediate solvent) for 2 × 10 min, incubated in propylene oxide/Spurr’s resin (1:1 mix) for 30 min, and in Spurr’s resin overnight. Sections were flat-embedded between two sheets of “Aclar” film and polymerized overnight at 60° Celsius.
Images were taken of each section and imported into Adobe Photoshop. Tracings were made of the BDA labeled axons present in each image. Then the tracings were aligned and collapsed into a single image so as to reveal the BDA labeled axons in the collection of sections. One section of the series was chosen for electron microscopic analysis, and a collection of bouton-like swellings on the regenerated axons were identified in advance. Ultrathin sections of 60 nm thickness were cut, mounted on copper grids and viewed using a JEOL 1400 electron microscope. Individual BDA-labeled boutons were then located and assessed at the electron microscopic level.
The selected sections were rinsed in 0.1 M cacodylate buffer and postfixed with 1 % osmium tetroxide in 0.1 M cacodylate buffer for 1 hour, rinsed in ddH20 for 2 × 10 min., dehydrated in increasing serial dilutions of ethanol (70%, 85%, 95%, 100% × 2) for 10 min each, put in propylene oxide (intermediate solvent) for 2 × 10 min, incubated in propylene oxide/Spurr’s resin (1:1 mix) for 30 min, and in Spurr’s resin overnight. Sections were flat-embedded between two sheets of “Aclar” film and polymerized overnight at 60° Celsius.
Images were taken of each section and imported into Adobe Photoshop. Tracings were made of the BDA labeled axons present in each image. Then the tracings were aligned and collapsed into a single image so as to reveal the BDA labeled axons in the collection of sections. One section of the series was chosen for electron microscopic analysis, and a collection of bouton-like swellings on the regenerated axons were identified in advance. Ultrathin sections of 60 nm thickness were cut, mounted on copper grids and viewed using a JEOL 1400 electron microscope. Individual BDA-labeled boutons were then located and assessed at the electron microscopic level.
