Reca 1

Frozen sections of cauda equine or fixed bEnd.3 cells were processed for immunostaining with antibodies against Jmjd3 (1:100, Abcam, Cambridge, MA, USA), ED-1 (CD68, 1:200; Serotec, Raleigh, NC, USA), RECA1 (1:100; Serotec, Raleigh, NC, USA), and ZO-1 (1:300, Invitrogen, Carlsbad, CA, USA) as previously described [19 (link)]. For double labeling, FITC or cy3-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA) were used. Additionally, nuclei were labeled with DAPI according to the protocol of the manufacturer (Molecular Probes, Eugene, OR, USA). In all controls, reaction to the substrate was absent if the primary antibody was omitted or if the primary antibody was replaced by a non-immune, control antibody. Serial sections were also stained for histological analysis with Cresyl violet acetate. Fluorescence labeled signal was detected by a fluorescence microscope (BX51, Olympus, Japan), and capture of images and measurement of signal colocalization was determined by MetaMorph software (Molecular Devices, Sunnyvale, CA, USA). To count the number of macrophages, eight sections were selected at 1-mm intervals in the compressed region of the cauda equina, and the total number of ED-1-positive cells per section was averaged.

Liver specimens were embedded in paraffin, cut into 5 μm sections, and stained with haematoxylin and eosin. The histological severity of I/R injury was graded using a modification of Suzuki’s criteria14 (link), as described in Table 1. The necrotic area was also quantitatively assessed using Image-Pro Plus v 6.0 (Media Cybernetics, Rockville, MD, USA). The results are presented as the percentage of necrotic area with respect to the entire area of the section. Immunostaining was performed for Ki-67 (BD Pharmingen, Franklin Lakes, NJ, USA) and cleaved caspase-3 (Cell Signaling Technology, Beverly, MA, USA) by staining with DAB followed by counter-staining with haematoxylin. The Ki-67 labelling index was determined as the percentage of Ki-67-positive hepatocytes per the total number of hepatocytes in 5 random visual fields (400×). Immunofluorescence was performed for rat endothelial cell antigen 1 (RECA-1) (Serotec, Oxford, UK).