Alpha smooth muscle actin

Immunohistochemistry was performed using a Ventana Bench Mark XT Autostainer (Ventana Medical Systems, Tucson, AZ, USA) and a LEICA BOND-III Autostainer (Leica Biosystems, Newcastle Upon Tyne, UK). Tested primary antibodies were as follows: NOVA1 (dilution 1:500; Abcam, Cambridge, UK), CD68 (dilution 1:150; DAKO), CD20 (dilution 1:1600; clone L26; DAKO), CD3 (dilution 1:200; LabVision, Fremont, CA, USA),
CD4 (dilution 1:200; Cell Marque, Rocklin, CA, USA), FOXP3 (dilution 1:100; Aviva Systems Biology, CA, USA), myeloperoxidase (dilution 1: 2000, DAKO), CD34 (dilution 1:50; clone QBEnd 10; DAKO), S100 (dilution 1:2000; DAKO), and alpha smooth muscle actin (dilution 1:500; clone 1A4; DAKO). Double staining with NOVA1/CD20, NOVA1/CD3, NOVA1/CD21, and NOVA1/CD68 was performed using palatine tonsil tissues for cellular localization of NOVA1 expression. Cell typing of immune cells and stromal spindle cells was also confirmed histomorphologically with H&E and immunohistochemical stains as follows: macrophages/monocytes/dendritic cells (CD68, S100, myeloperoxidase), B-lymphocytes (CD20), T-lymphocytes (CD3), regulatory T cells (FOXP3), neutrophils (myeloperoxidase), stromal spindle cells (Schwann cells, fibroblasts, support cells, and endothelial cells; S100, smooth muscle actin, and CD34).

Samples of arterial wall and intra-luminal thrombus obtained from AAA patients were embedded in paraffin, and 4 μm cross-sections were cut. Ceroids were detected by direct observation of tissue by fluorescence microscopy (ceroids autofluoresce at 550 nm, producing a red signal). Immunohistochemistry was performed with antibodies against the following proteins: PRDX6 (ab16947, abcam), the lipid peroxidation marker MDA (ab6463, abcam), the neutrophil marker CD15 (Dako), and alpha smooth muscle actin (Dako). Sections were then incubated with the appropriate biotinylated secondary antibody and ABComplex, followed by staining with 3,30-diaminobenzidine (DAB), hematoxylin counterstaining, and mounting in DPX medium.