Ema clone e29

Immunostaining was performed using a routine indirect peroxidase method. The following antibodies were applied: TP53 (Dako, Denmark), Ki-67 (Dako), and CD24 (clone 24C02, Dianova, Hamburg, Germany). These antibodies were used at a final concentration of 1–2 μg/ml. For immunohistochemical detection of osteopontin and osteonectin, deparaffinized and ethanol-dehydrated tissue sections were incubated overnight with polyclonal rabbit antibodies to osteonectin (dilution 1:1,000) and osteopontin (dilution 1:3,500) at room temperature. The antibodies were kindly provided by L.W. Fisher, NIH, USA (13 (link)). Sections were then incubated with a monoclonal mouse-anti-rabbit antibody (Dako, Glostrup, Denmark) for 30 min followed by signal detection using the Dako ChemMate APAAP system and the Dako TechMate^™ 500 plus automatic stainer.
FACS analysis was performed according to protocols described previously (14 (link)). The following antibodies were applied: CD24 (clone 24C02), CD20 (clone L26, Dako), EMA (clone E29, Dako) and rabbit anti-mouse immunoglobulins (code no. F0313, Dako).

Immunocytochemical analysis was possible only on CBs, on three micron-thick unstained sections. An initial panel of antibodies was used in malignant cases to differentiate lymphoma from carcinoma and sarcoma, namely pan-cytokeratin (clone AE1/3; DAKO, Carpinteria, CA, USA), CD45/LCA (Clones 2B11+PD7/26; DAKO), CD20 (clone L26; DAKO), CD79α (clone JCB 117; DAKO), CD3 (polyclonal; DAKO), EMA (clone E29; DAKO), CD30 (clone Ber-H2; DAKO), CD15 (clone Carb-3; DAKO) and PAX5 (clone DAK-PAX5; DAKO). In case a diagnosis of B-lineage NHL had been advanced, a second lineage of immunocytochemical markers was performed, including BCL2 (clone 124; DAKO), BCL6 (clone PG-B6p; DAKO), CD10 (clone 56C6; DAKO), CD5 (clone 4C7; DAKO), CyclinD1 (clone EP12; DAKO), MUM1/IRF4 (clone MUM1p; DAKO) and Mib1 (clone Ki67; DAKO). All staining procedures were carried out on Autostainer Link48 (DAKO, DAKO), according to the manufacturer’s instructions and in the presence of appropriate controls. Neither morphology nor first- nor second-line immunocytochemical analyses aroused suspicion of T-lineage NHL.

Immunohistochemistry was performed on a Benchmark Ultra (Ventana, Tucson, AZ) automated stainer using the CC1 mild protocol for antigen retrieval and monoclonal antibodies against E-cadherin (clone ECH-6, Zytomed Systems, Berlin, Germany, 1:100), β-catenin (clone 14, Beckton Dickinson, Heidelberg, Germany, 1:100), CK7 (clone OV-TL12/30, DAKO, Glostrup, Denmark, 1:100), Epithelial membrane antibody (EMA, clone E29, DAKO, 1:600), ER (clone SP1, Ventana, undiluted ready-to-use), BCL2 (clone 124, DAKO, 1:100), GATA binding protein 3 (GATA3, clone L50-823, BioCare Medical, Concord, CA, 1:200), mammaglobin (MGBN, clone 304-1A5, Biologo, Kiel, Germany, 1:10) and v-erb-b2 avian erythroblastic leukemia viral oncogene homolog 2 (ERBB2, clone 4B5, Ventana, undiluted ready-to-use). Detection of the immune reaction was achieved with the ultraView DAB kit (Ventana).