Frozen sections were cut at a thickness of 5 μm and mounted onto microscope slides. Sequential sections were used for a single antibody or for two antibodies (co-expression- as specified in Tables 1, 2) including for the negative controls. Samples were fixed with acetone for 15 min. at 4°C and washed with PBS. Then, incubated with the primary antibodies diluted at 1:100 in blocking buffer of 10% normal goat serum in RPMI-1640 medium, overnight at 4°C (2 (link)). The following cellular markers were used to identify and quantify cell populations in the carotid plaques; PMNs were identified by primary antibodies for CD66b, NE, and MPO and macrophages were identified by CD163. Double staining of CD66b(mono)/CD163(poly) was performed to identify potential co-expression. Additional markers included the scavenger receptors CD36 and CD68 for foam cells, the oxidative stress marker 3-NT, hypoxia inducible factor 1α (HIF-1α), VEGF, CD31 – for vessel identification by the presence of endothelial cells, and smooth muscle cell actin (SMC-actin), a marker of arterial wall remodeling.
After overnight incubation with primary antibodues the slides were washed and incubated with 1/400 secondary antibodies in blocking buffer, at room temperature, for 40 min. Secondary antibodies included Cy2 (CF 488A)-conjugated goat anti-rabbit IgG and/or Cy5 (CF 647)-conjugated goat anti-mouse IgG (Biotium, Hayward, CA). Isotype controls included: purified mouse IgG1 (clone MOPC-21, BioLegend, San Diego, CA), and normal rabbit IgG (sc-2027, Santa Cruz Biotechnologies, Santa Cruz, CA). After 40 min. incubation, slides were washed and mounted with mounting medium containing 4’, 6-diamidino-2-phenylindole (DAPI) for nuclear staining (Vectashield H-1000, Vector lab. Inc. Burlingame, CA).
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