Anti bcl6

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.

De-identified primary FL specimen tissues were obtained in accordance with the guidelines and approval of the Institutional Review Board of the Weill Cornell Medical College and in accordance with the Helsinki protocols. We selected the specimens based on estimated tumor content >80% by our collaborating pathologist Dr. Wayne Tam. Single-cell suspensions from lymph node biopsies were obtained by physical disruption of tissues (using scalpels and cell strainers), followed by cell density gradient separation (Fico/Lite LymphoH; Atlanta Biologicals, Norcross, GA). Cell number and viability were determined by trypan blue dye exclusion, and cells were cultivated in medium containing 80% RPMI and 20% human serum supplemented with antibiotics, L-glutamine 4 mM and HEPES 10 mM. Cells were exposed in duplicates to control and RI-BPI at indicated concentrations for 48 h. Viability was determined as detailed above. The BCL6 protein status was determined in paraffin-embedded samples by immunohistochemistry using anti-BCL6 (Dako North America, Carpinteria, CA).