Characterization of human primary glioma surgical samples. Extensive GFAP (A, left panel) expression was detected in primary glioma surgical sample frozen sections using immunofluorescent staining with a rabbit anti-GFAP antibody. (B, left panel) Numerous dividing cells were detected in the same surgical glioma sample using an anti-Ki67 antibody. Right panels in (A) and (B) are nuclear counterstaining for GFAP and Ki67 stained sections, respectively. (C) Demonstration of specificity of anti-L1 antibodies anti-cytoplasmic polyclonal NCAM-L1 (left panel) and anti-ectodomain monoclonal UJ127 (right panel) by western blot analysis. Human L1-expressing quail QT6 cells (QT6/hFL1) were used as positive controls (PC), untransfected QT6 cells were used as negative controls (NC), and plain QT6 cell culture media (M) was used as an additional negative control. (D) L1 expression was found in human primary gliomas surgical samples (sample numbers 10-15 and 17-20) by western blot analysis using UJ127 anti-L1 antibody. Transfected QT6/hFL1 cells were used as positive control and QT6 cells were used as a negative control. Gels were loaded with 10 μg total protein and probed for GAPDH as a loading control (see text). (E) Analysis of surgical samples 18-20 using anti-L1 antibody NCAM-L1. Same blot was used as for (D, right panel, 10 μg total protein/well). GAPDH was used as a loading control. (F) Analysis of glioma surgical samples for L1 protease ADAM10, revealing that all samples were positive predominantly for active ADAM10 (approx. 55 kDa). (G) Surgical samples # 7, 18, 19, and 20 were analyzed by western blot with a rabbit anti-NF-M antibody to detect neurofilament expression. Adult rat brain (RB) lysate was used as a positive control for NF-M staining. (H) Media from surgical sample cells grown in culture were analyzed by western blot for L1. Soluble L1 was detectable in media from sample # 21. Positive controls (PC) were cell lysates from QT6/hFL1 cells, and untransfected cell lysate was used as negative control (NC). Media from CHO cells transfected with an L1 ectodomain vector (CHO-L1ecto) were also used as a positive control for soluble L1. Media from untransfected CHO cells were used as a negative control.