Ed 1 cd68

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.

Cells in single-cell suspension were added to 96-well V-bottom polypropylene plates (BD Falcon) at 10⁶ cells/well and incubated with saturating concentrations of mAbs (see below). Cells were washed with FACS buffer and incubated in BD Cytofix/Cytoperm for 20 mins at room temperature and washed twice in BD Perm/Wash before staining with Abs to CD68. Fluorescence minus one control were used in all experiments. Dead cells were stained with Live/Dead Violet (Invitrogen) after washing the cells twice in Dulbecco’s PBS without sodium azide and FCS. A SORP BD LSR II analytic flow cytometer (BD Biosciences) was used for acquisition and the data were analyzed with FlowJo (Tree Star). The following Alexa Fluor 488–, PE-, PerCP-Cy5.5–, allophycocyanin-, allophycocyanin-Cy7–, Qdot-655–, and biotin-conjugated Abs were used for flow cytometry: 2.4G2/BD Fc Block (CD32), OX-8 (CD8a), OX-1 (CD-45), HRL-1 (CD62l) and WT.5 (CD11b) were purchased from BD Pharmingen (San Diego, CA); R73 (αβTCR), OX-62 (CD103), W3/25 (CD4), 24F (CD86), 3H5 (CD80) purchased from BioLegend (San Diego, CA USA); OX-39 (CD25), OX-17 (RT1-D), His24 (CD45R/B220), His36 (ED-2) were purchased from eBioscience (San Diego, CA); and ED1 (CD68), purchased from AbD Serotec.