Depletion of B-cell subsets in the lymphoid compartment. (A) Immunohistochemistry of B-cell depletion in the spleen and mesenteric and inguinal lymph nodes. Transgenic hCD20 mice (BALB/c) received 250 μg of antihuman CD20 mAb or an isotype-matched control mAb (WR17) intravenously and killed on days 2 or 7. Tissues were harvested, embedded, and frozen, and sections stained for B cells (B220; red) and T cells (CD3; green). Scale bar represents 500 μm. Slides were viewed with a Leica SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany) using a 10× dry plan fluotar lens with a 1.5× optical zoom with Vectashield mount (Vector Labs, Burlingame, CA). Images were acquired using Leica Confocal Software v2.61 and were processed with Adobe Photoshop version CS2 software (Adobe Systems). (B) Mice were treated for 2 or 7 days as indicated in panel A, then cells isolated from the bone marrow (BM), spleen (Spl), or lymph node (LN). The proportion of B cells remaining in these organs was determined by flow cytometry, compared with controls and expressed as a percentage. Bars represent mean plus or minus SEM (n = 3 from 2 independent experiments).