Colocalization of MMP-9 with CD44v and α4β1 integrin in B-CLL cells. B-CLL cells from a representative sample were added to glass coverslips coated with 5 μg/mL poly-lysine. After 1 hour at 37°C, cells were fixed and analyzed by confocal microscopy using specific Abs for α4, β1, CD44v7,8, and CD44, followed by Texas-Red–labeled secondary Abs. MMP-9 was detected with specific pAbs and Alexa 488–labeled secondary Abs. Colocalization of MMP-9 with CD44v and α4β1 was further demonstrated using dot-plot analyses. Images were acquired using a confocal scanning inverted AOBS/SP2 microscope (Leica Microsystems, Heidelberg, Germany) with a 63×/1.3 NA PL-APO glycerol immersion objective. Leica's LCS 15.37 dye-separation software was used for colocalization studies; when necessary, Adobe Photoshop 7.0 (Adobe Systems, San Jose, CA) was used for image processing. Bar represents 4 μm.