Resin-embedded sections tissue dehydration

After the last experiments, the rats are euthanized by injection of an overdose of pentobarbital and then perfused intracardially with a phosphate-buffered 2.0% paraformadehyde - 2.5% glutaraldehyde fixative. Methyl green solution was injected to confirm the location of the catheter after the perfusion. The spinal cord and nerve roots were dissected out and immersed in the same fixative for 4 h. Two specimens (10 mm rostral and caudal to the conus medullaris from each rat were postfixed with cacodylate-buffered 1 % osmium tetroxide dehydrated in a series of graded alcohol solutions, and embedded in epoxy resin. From the embedded tissue, 1-pm transverse sections were obtained and stained with toluidine blue dyes. Sections obtained from 10 mm rostral to the conus (caudal spinal cord) were used for qualitative evaluation. Quantitative analysis of nerve injury was performed using the sections obtained form 10 mm caudal to the conus. Each fascicle present in the cross section was assigned to an injury score 0 to 3. The injury score for each cross section was then calculated as the average score of all fascicles present in the cross section. 

For LM and TEM, the treated apical meristems were fixed frozen in 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.2 at 4°C overnight. Control unfrozen samples were also fixed in the same way. For TEM, tissues were postfixed in 1% OSO4 in water for 2 h, dehydrated in a graded ethanol-acetone series and embedded in low-viscosity Spurr s resin. Sections were stained with uranyl acetate followed by lead citrate and examined in a Zeiss EM109T transmission electron microscope. For LM, sections were stained with Sudan Black B (Sigma S 2380 Cl 26150) and Toluidine blue O (Sigma T 3260 Cl 52040). 

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