Mouse anti human ige

Immunohistochemistry with mouse anti-human IgE (1:70, Southern Biotech, Birmingham, AL) and rabbit anti-human FcεR1 (1:300; Santa Cruz Biotechnology) and their colocalization with apoptosis (TUNEL, Apoptosis Tag kit; Millipore) was performed on acetone fixed frozen sections from human AAA (n = 11) and control normal aortas (n = 5) by avidin/biotin method. Specimens obtained by protocols approved by the Human Investigation Review Committee at the Brigham and Women's Hospital were immediately immersed in saline, embedded in OCT, and stored at −80°C freezer. To localize IgE and FcεR1 to vascular SMCs, ECs, and CD4⁺ T cells, we performed double immunofluorescent staining (n = 11) with mouse anti-IgE (1:10) or rabbit anti-FcεR1 (1:80) antibodies incubated overnight, followed by anti-mouse or anti-rabbit Alexa-555 (Invitrogen, Grand Island, NY). Sections were washed, blocked with 4% of appropriate normal serum, and incubated with mouse anti-α-actin (1:40, HHF35; Enzo-Diagnostics, Syosset, NY), CD31 (1:40, Dako, Carpinteria, CA), and CD4 (1:20, Dako), followed by anti-mouse Alexa-488 (Invitrogen).

Mouse anti-human IgG₁ (#9054-01), mouse anti-human IgG₂ (#31-7-4), mouse anti-human IgG₄ (#9200-01), and mouse anti-human IgE (#9250-01) were obtained from Southern Biotechnology (Birmingham, AL). Mouse anti-human IgG₃ (#05-3600) was obtained from Invitrogen (Carlsbad, CA) and mouse anti-human IgM (#555856) and mouse anti-human IgA (#555886) were obtained from BD Biosciences (San Jose, CA). Secondary detection reagents including biotin-mouse anti-human kappa light chain (#555790) and biotin-mouse anti-human lambda light chain (#555794) were obtained from BD Biosciences (San Jose, CA). R-Phycoerythrin-conjugated streptavidin (#016-110-084) and R-Phycoerythrin-conjugated donkey F(ab’)₂ anti-mouse IgG (H + L) (#715–116-150) were obtained from Jackson ImmunoResearch (West Grove, PA).