Real detection system alkaline phosphatase red

CEA samples were divided into three parts: (i) areas with massive plaque formation (apart from the plaque core, usually completely calcified and not evaluable), (ii) areas adjacent to the plaque corresponding to (iia) internal carotid artery and to (iib) common carotid artery, where the plaque was almost or completely absent. The segments were designated for both histology and immunohistochemistry and then embedded in paraffin. Some sections were stained with hematoxylin/eosin and analyzed using an optical microscope. Other sections were used for immunohistochemistry: sections were deparaffinized in xylene and grades of alcohol, then rehydrated in water. Antigen retrieval was performed by heating in a pressure cooker with EDTA for 15min. After a washing in TRIS buffered saline/Triton X-100 solution, sections were incubated in a blocking solution (PBS, bovine serum albumin [BSA], NaN₃) for 10 min and then incubated overnight at 4°C with mouse anti-human CD66b antibody (1:50). Dako REAL Detection System/Alkaline Phosphatase/RED was used as revelation system. Negative control was obtained omitting primary antibody. All the sections were analyzed by means of light microscopy and evaluated by a semiquantitative method.

Paraffin sections (5 µm) of a FACAS patient skin sample and control skin were prepared as explained above, and processed for routine (hematoxylin/eosin) and immunohistological stainings. We used an immunohistochemistry protocol (streptavidin–biotin labeling) employing primary antibodies targeting MPO (MAB3174, R&D Systems) 1:400 overnight at 4 °C or CD163 (ab189915, Abcam) 1:400 overnight at 4 °C. After washing 3 × 5 min with TBS, REAL Detection System Alkaline Phosphatase/RED (K5005, Dako) for MPO staining or labeled polymer-HRP anti-rabbit (K4011, Dako) together with AEC + high sensitivity substrate chromogen ready-to-use (K3461, Dako) for CD163 staining were applied according to the manufacturer’s instructions, and subsequently analyzed by bright-field microscopy. Samples of tonsil tissue served as positive controls. Sections omitting the primary antibody served as negative controls.