Anti cd3

Thirty μm-thick coronal floating sections were incubated with primary antibody overnight at 4°C to detect neuroblasts using anti-doublecortin (DCX) (1:4000, Abcam, Cambridge, United Kingdom); microglial cells using anti-Iba1 (1:200, Wako, Japan), T-lymphocytes using anti-CD3 (1/200, Dako, Santa Clara, United States), astrocytes using anti-GFAP (1:200, Millipore, Burlington, MA, United States) and vessels using the anti-glucose transporter-1 (Glut-1) (1:500, Millipore, Burlington, MA, United States). Appropriate fluorescent-labeled secondary alexa fluor 594 or 488 antibodies (Molecular Probes, Eugene, OR, United States 1:400) were applied for 1 h at room temperature. Specificity was checked by omitting the primary antibody. As doublecortin is expressed in newborn and migrating neurons, we assumed that DCX+ cells reflect neurogenesis (Brown et al., 2003 (link); Jin et al., 2010 (link)).

Tumors were harvested at an average tumor size of 150-200 mm³ and snap frozen. Tissues were cryosectioned at 8 µm and immunohistochemical (IHC) experiments were conducted as follows: slides were fixed in cold acetone or 4% formalin, blocked in 5% normal mouse serum (Jackson Immunoresearch) and incubated with primary antibodies for 1.5 h at room temperature. Primary antibodies were anti-CD3 (Dako, A0452, #280), anti-CD4 (Affymetrix, #14-0042) and anti-CD8a (Affymetrix, #14-0081). Sections were dried and stained with haematoxylin/eosin in a Leica ST4040 automatic stainer.
Assessment of T cell infiltration was performed in a blinded fashion and scored as 0 (negative), 1 (<150 cells per mm²), 1-2 (150-300 cells per mm²), 2 (300-500 cells per mm², 2-3 (500-800 cells per mm²), and 3 (>800 cells per mm²⁾. N=3 per syngeneic tumor model.

Three samples were isolated from each patient: macroscopically normal abdominal aorta, incipient ASVD lesion, and a complicated lesion. Sections were H/E-stained and classified according to the modified classification of American Heart Association (AHA) [6 (link)]. Formalin-fixed, paraffin-embedded aortas, were sliced at 4 μm and stained with polyclonal anti-CD3, anti-S100 (Dako, Glostrup, Denmark), anti-CD40, anti-CD40L (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-NF-κB-p65 (phosphor S536) (Abcam, Cambridge, UK) antibodies. Sections were counterstained with hematoxylin to make nuclei evident. Human parotid glands or ganglions were used as positive controls. Isotype controls were performed using antibody buffer supplemented with irrelevant immunoglobulines of the same isotype, species and concentration as the primary antibody (ThermoFisher, Rockford, IL USA). Negative controls were performed by omitting primary antibodies. Positively stained cells were counted at x100 to x200 and acquired with a digital camera. The percentage of positive cells was assessed irrespective of the staining intensity. Results were expressed as the percentage of each population regarding the total number of cells in the intima or adventitia.