Purinergic Signalling (2008) 4:61–71 baseline behavioural and histological analysis. The other rats received either daily intraperitoneal (i.p.) injections of 8 mg/kg guanosine or the same volume of saline containing 0.001 N NaOH [31] for 2 weeks. The open field walking testing was performed once a day from days 35 to 48 post- surgery (0 to 14 days post-treatment). Groups of 9–12 rats were killed by perfusion/fixation for immunohistochemical analysis at 1, 3, 7 or 14 days after treatment. BrdU administration Bromodeoxyuridine (BrdU) was used to label proliferating cells after treatment. BrdU (50 mg/kg in sterile saline; Sigma, B-5002) was injected intraperito- neally daily for 1, 3, 7 or 14 consecutive days, beginning the day treatment was initiated, thereby reflecting cell proliferation over the entire treatment period. Tissue processing and immunohistochemistry On post- operative days 35, 36, 38, 42 or 49 (representing baseline, 1, 3, 7 or 14 days treatment), rats were deeply anaesthetized with sodium pentobarbital (50–60 mg/kg body weight, i.p.) immediately following the final locomotor testing and perfused transcardially with 100 ml 0.1 M phosphate- buffered saline (PBS) containing 0.1% heparin, followed by 300–500 ml of 4% paraformaldehyde (PFA). T9 to L1 segments of the spinal cords were removed and incubated in the same fixative for 2 h at room temperature and then cryoprotected in 30% sucrose PBS solution. A segment of each cord, extending from 5 mm rostral to 5 mm caudal to the lesion site was embedded in medium (Tissue-Tek® O.C.T. compound, Sakura Finetek USA, Inc., Torrance, CA, USA). Serial transverse sections were cut at 15- to 20-μm intervals on a cryostat and mounted onto slides (ColorFrost/Plus; Fisher, Pittsburgh, PA, USA). Every fifth section was stained with the lipophilic dye Luxol fast blue to stain myelin [38]. Adjacent sections were stained immunohistochemically with the various antibodies described below. For BrdU immunohistochemistry, 6% hydrogen peroxide was used to reduce endogenous peroxidase activity, followed by three 10-min PBS rinses and incubation in 1 N HCl at 37°C for 30 min. After rinsing, a blocking solution [5% bovine serum albumin (BSA) in 0.1% Triton- 100] was applied for 1 h; sections were subsequently incubated in rat anti-BrdU (1:200 in 1% BSA in 0.1% Triton-100; Serotec, Raleigh, NC, USA) at 4°C overnight in a humid chamber. Sections were then rinsed and incubated with biotinylated horse anti-rat IgG secondary antibodies. Sections were then incubated with Elite ABC (Vector Laboratories, Burlingame, CA, USA) for 1 h, followed by 3,3′-diaminobenzidine (DAB) substrate kit for peroxidase (Vector Laboratories, Burlingame, CA, USA) for 5–10 min. Sections were dehydrated, coverslipped and examined under a microscope. 63 To determine the fate of newly born cells after SCI, double-fluorescent immunolabelling was performed, com- bining BrdU with one of the cell-specific phenotypic markers listed below. For double immunofluorescence, sections were pretreated with HCl as described above and incubated with BrdU antibody mixed with one of the following antibodies in PBS at 4°C overnight in a humid chamber: rabbit anti-MBP (myelin basic protein) polyclonal antibodies (1:50; Chemicon Int., Temecula, CA, USA), rabbit anti-NG2 polyclonal antibodies (chondroitin sulfate proteoglycan for oligodendroglial progenitors; 1:200; Chemicon Int. Temecula, CA, USA), mouse anti-Rip monoclonal antibodies (mature oligodendrocyte-specific marker: Reactive Immunology Protein; 1:200; Chemicon Int., Temecula, CA, USA; [39]), rabbit anti-glial fibrillary acidic protein (GFAP) polyclonal antibodies (1:600; Zymed® Lab-SA System Kit, San Francisco, CA, USA) and mouse anti-microglial-specific marker OX-42 mono- clonal antibodies (1:200; CD11b, Research Diagnostics Inc., Flanders, NJ, USA). For Rip and OX-42 monoclonal antibodies single staining, sections were developed with fluorescein isothiocyanate (FITC)-conjugated goat anti- mouse IgG. For BrdU and polyclonal antibodies (NG2 or MBP or GFAP) double immunolabelling, sections were developed using a mixture of FITC-conjugated goat anti-rat IgG and rhodamine-conjugated goat anti-rabbit IgG in 1% normal goat serum and 0.25% Triton X-100; 1:200 (Invitrogen, Carlsbad, CA, USA) for 2 h. To examine the contribution of Schwann cells from the peripheral nervous system (PNS) in the remyelination process, sections were immunolabelled with specific markers for Schwann cell myelin P0 (a rabbit polyclonal anti-P0 antibody was kindly donated by Prof. Marie T. Filbin, Department of Biology, CUNY, NY, USA; 1:200) and secondary antibody using rhodamine-conjugated goat anti-rabbit IgG. For detection of apoptotic cells, a terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) stain was performed using the ‘In situ Cell Death Detection Kit, Fluorescein’ (Roche Molecular Biochemicals, Mannheim, Germany), according to the manufacturer’s instructions. Quantification To quantify the amount of myelin present at the injury site before and after treatment, the Luxol fast blue- stained section from each cord (n= 7–10 for each group) that contained the greatest lesion area was selected together with the adjacent two caudal and two rostral Luxol blue-stained sections. Luxol fast blue staining was also done on cords of five additional unoperated normal animals for comparative purposes. Digital photographs were taken of the sections. The total area of each section and the area of the fast blue- stained portion were measured using a computerised Bioquant BQ-TCW98 image analysis program by an investigator who was blind to group assignment.