Anti cd3

The glioma core was examined in different tissue from that used for electro-physiological recording to establish the type and grade of the tumor according to the 2007 WHO classification (33 ). Tissue was fixed in paraformaldehyde 4 % in PBS for 48-72 h. Paraffin-embedded tissue sections (20μm) were stained by Hematoxylin-eosin or processed for immunohistochemistry. After deparaffinization and inhibition of endogenous peroxidase, sections were microwaved in a sodium citrate buffer pH=6 for KCC2 antigen retrieval. The following antibodies were used: anti CD3 (Ventana); anti CD20 L26 1/1000 (Dako); anti CD68 KP1 1/1000 (Dako); anti KCC2 1/2000 (gift from Kai Kaila); anti Ki67 1/50 (Dako); anti NeuN 1/500 (Chemicon). We compared tumoral and peritumoral areas using a cut off (10 or more tumor cells per high power field (HPF); x400, 0.2 mm²) to distinguish regions of tumor infiltration and regions containing isolated tumor cells. CD68 immunoreactive cells were counted per HPF and Ki67 immunoreactive cells were counted per 10 HPF. At least 100 neurons were counted to determine the pattern of KCC2 immunoexpression.
On histopathological examination, we defined areas with solid tumor mass, high tumor infiltration and low tumor infiltration by isolated glioma cells in 18 slices taken adjacent to a recorded slice (10 patients, 4 high-grade, 6 low-grade gliomas).

Charged slides from a subset of primary melanomas were deparaffinized in xylene, rehydrated in ethanol, and heated in EDTA pH 9.0 for antigen retrieval. Slides were then co-stained with anti-CD2 (pre-diluted), anti-CD3 (pre-diluted), and anti-CD56 (pre-diluted) monoclonal antibodies (Ventana Medical Systems, Oro Valley, AZ) and anti-FoxP3 monoclonal antibodies (Abcam, Cambridge, MA). Staining was visualized using fluorochrome-conjugated secondary antibodies (Invitrogen, Grand Island, NY). Slides were sealed using fluorescence mounting medium (Dako, Glostrup, Denmark). Slides were visualized using a Nikon A1 confocal microscope and NIS-Elements software at the Confocal and Specialized Microscopy Facility of Herbert Irving Comprehensive Cancer Center at Columbia University.

Hematoxylin and eosin stained lymph node sections were analyzed at low-power (12.5X) on a DM4000B microscope (Leica Biosystems), to capture the nodes entire two-dimensional area with a Spot RT/SE Slider camera (Spot Imaging). Follicular structures were traced and scored for circularity in silico with ImageJ software (NIH freeware) using the formula:
circularity=4π(area/perimeter²)
Lymph node follicular composition was determined by dividing the combined 2-dimensional areas of all follicular structures by the total lymph node area excluding adipose rich or acellular fibrous tissues. All pathology images were reviewed by one or more hematopathologists. Immunohistochemical staining was performed with anti-CD21 (1:100, Dako), anti-CD3 (pre-diluted, Ventana), anti-CD20 (1:200, Dako) and anti-BCL6 (1:50, Dako) antibodies.