Rabbit anti cd45

The slides of passage 3 BMSCs were fixed with 4% paraformaldehyde (Sinopharm, China) for 15 min and then rinsed 3 times using PBS. Afterwards, the cell slides were ventilated with 0.5% Triton X-100 (Beyotime, China) at room temperature for 20 min and blocked with goat serum for 30 min. A PBS wash followed each step. Diluted primary antibodies were added to the slides and coincubated at 4°C overnight. The primary antibodies used for immunostaining included mouse anti-CD90 (1/500, Abcam), mouse anti-CD11b (1/200, Abcam), mouse anti-CD44 (1/500, Abcam), mouse anti-CD29 (1/200, Abcam), rabbit anti-CD45 (1/100, Abcam), and rabbit anti-CD271 (1/500, Abcam). After 3 rinses with PBS, the slides were coincubated with diluted secondary primary antibodies (DyLight® 488-goat anti-rat IgG, 1/100, Invitrogen; DyLight® 488-goat anti-rabbit IgG, 1/100, Invitrogen) for 1 h. Following 3 times of PBS wash, the slides were coincubated with 4′,6-diamidino-2-phenylindole (DAPI) avoiding light for 5 min. After 4 subsequent PBS washes, the slides were dried using absorbent paper and mounted with an antifluorescence quenching agent (Southern Biotech, Alabama, US). Images were collected using a fluorescence microscope (Olympus BX53, Japan).

PFA-fixed tissue sections were rinsed in PBS with 1% Triton-X100. For antigen activation, 0.1% trypsin in PBS was added to the tissue sections. After washing in PBS, the tissue sections were blocked with Blocking One Histo. The sections were incubated with the appropriate primary antibody overnight at 4°C. The histological results from the aortic wall were assessed after staining using the following antibodies as described above. CD45 and CD105 in the aortic wall were assessed after staining using the following antibodies: rabbit anti-CD45 (1:50; abcam, Cambridge, UK) and mouse anti-CD105 (1:50; abcam, Cambridge, UK). The antigen was detected with anti-rabbit or anti-mouse Alexa488-, and anti-rabbit or anti-goat Alexa549-conjugated secondary antibody (Rockland Immunochemicals, Limerick, PA, USA). Sections were subjected by DAPI solution (KPL, Gaithersburg, MD, USA) for nuclear staining for 5 minutes. Slides were covered with Fluoromount^TM (Diagnostic BioSystems, Pleasanton, CA, USA). Sections of negative control were not subjected to the primary antibodies, and representative images of negative control were shown in Figure S15.

Spinal cords were dissected from the spinal columns and paraffin sections from the lumbar region were prepared and analyzed for changes in the expression of CD45, CD3, and myelin basic protein (MBP) using multi-channel fluorescence immunohistochemistry using immunostaining methods as previously described.^{50 (link)} Rabbit-anti CD45 (1:50, Abcam), rat anti-CD3 (1:100, Abcam) and mouse anti-MBP (1:1000; Covance) and appropriate fluoroconjugated secondary antibodies (Alexa 546 or Alexa 488, Molecular Probes, Inc.) were used. Cell nuclei were visualized by staining with DAPI (1:5000, Molecular Probes, Inc.). Sections were photographed with an Axioskop 40 fluorescence microscope (Zeiss) and images processed under identical conditions using Image J (version 1.53t).