Immunohistochemical Analysis of Atherosclerotic Plaques

Frozen sections were cut at a 5 μm thickness and mounted on microscope slides. The 5-μm-thick sections were stained with hematoxylin and eosin (H&E), and lipid deposits in the plaques were visualized by Oil Red O staining as previously described (3 (link), 15 (link)). Primary antibodies against CD66b, CD163, and CD68 cellular markets were diluted to 1:100, and a Histostain-Plus Kit AEC, Broad Spectrum (Invitrogen) was used for their detection. The sections were incubated with the primary antibodies for 2 hrs. at 37°C. Then, the sections were incubated with secondary antibody from the Histostain-Plus kit, for 30 min at 37°C. The 3-amino-9-ethylcarbazole (AEC) was used as a chromogen to detect the antibodies according to manufacturer’s instructions. Polymorphonuclear neutrophils (PMNs) were identified by the expression of CD66b, macrophages were identified by the expression of CD163 and foam cells were identified by the expression of CD68 scavenger receptors. Isotype controls were used as specified in the list of antibodies. Lipid deposits were stained with Oil Red O. Sequential sections were stained each for an antibody including for negative controls. At least 3 different sections were cut from the center of each plaque for each CD marker and in each section at least 5 different fields were analyzed.
Collagen and non-collagen proteins were detected by differential staining in tissue sections with two dyes - Sirius Red for all collagens and Fast Green for non-collagen proteins.