Immunofluorescence staining of sections from small (unruptured) and giant (ruptured) IA samples collected in surgery followed a previously established protocol.
41 (link) Briefly, fresh IA domes were fixed in 4% paraformaldehyde and dehydrated in graded methanol overnight at −20 °C. Then, samples were rehydrated, washed, and blocked with SEA BLOCK Blocking Buffer sequentially, followed by incubation with primary antibodies overnight at 4 °C. anti‐CD31 (1:3000; HUABIO, Anhui, China), anti–α‐smooth muscle actin (1:4000; HUABIO), anti‐nerve/glial antigen‐2 (1:100; Oasis), anti‐CD11b (1:800; HUABIO), anti‐CD68 (1:2000; HUABIO), anti‐CD163 (1:2000; HUABIO), anti‐CD86 (1:2000; HUABIO), and anti‐CD3 (1:800; HUABIO) were employed to denote endothelial cells, SMCs, pericytes, neutrophils, monocytes/macrocytes, and T lymphocytes, respectively. The second antibodies used were mouse or rabbit poly‐HRPs, labeled with cyclic‐480, cyclic‐550, and cyclic‐630 dyes using IRISKit HyperView mIF kit (LUMINIRIS, China). DAPI was used as the nuclear counterstain. Tissue section staining was done by Lumin Iris Bio Tech Co. Ltd. (Chengdu, China). The slices were scanned using the Olympus VS200 scanner. The images were adjusted using ImageJ followed by analyzing using the QuPath software version 0.4.4.
41 (link) Briefly, fresh IA domes were fixed in 4% paraformaldehyde and dehydrated in graded methanol overnight at −20 °C. Then, samples were rehydrated, washed, and blocked with SEA BLOCK Blocking Buffer sequentially, followed by incubation with primary antibodies overnight at 4 °C. anti‐CD31 (1:3000; HUABIO, Anhui, China), anti–α‐smooth muscle actin (1:4000; HUABIO), anti‐nerve/glial antigen‐2 (1:100; Oasis), anti‐CD11b (1:800; HUABIO), anti‐CD68 (1:2000; HUABIO), anti‐CD163 (1:2000; HUABIO), anti‐CD86 (1:2000; HUABIO), and anti‐CD3 (1:800; HUABIO) were employed to denote endothelial cells, SMCs, pericytes, neutrophils, monocytes/macrocytes, and T lymphocytes, respectively. The second antibodies used were mouse or rabbit poly‐HRPs, labeled with cyclic‐480, cyclic‐550, and cyclic‐630 dyes using IRISKit HyperView mIF kit (LUMINIRIS, China). DAPI was used as the nuclear counterstain. Tissue section staining was done by Lumin Iris Bio Tech Co. Ltd. (Chengdu, China). The slices were scanned using the Olympus VS200 scanner. The images were adjusted using ImageJ followed by analyzing using the QuPath software version 0.4.4.