β₁-integrin and BMP4 expression in BM and spleen. (A) Prominent labeling of endothelial cells and hematopoietic cells, including megakaryocytes, is seen in the normal tibia using anti-β₁, but labeling of only endothelial cells is seen (B) in β₁^Δ/Δ tibia. (C) Labeling of endothelial cells and megakaryocytes with 9EG7 antibody (reacting with activated β₁) in normal tibia. (D) Labeling only of endothelial cells in β₁^Δ/Δ tibia. (E) Splenic sections from normal mice after PHZ labeled for F4/80 (diaminobenzidine method) for macrophages and counterstained with hematoxylin. Note the abundance of small dark nuclei (erythroid cells). (F) Splenic sections from β₁^Δ/Δ mice after PHZ. Fewer erythroid cells together with many unengaged macrophages. (G) Splenic sections from normal mice labeled with 9EG7 antibody (activated β₁). Prominent labeling of endothelial cells is seen. (H) Splenic sections from β₁^Δ/Δ mice similarly labeled with 9EG7. (I) BMP4 labeling of normal spleen after PHZ (day 6) and (J) of β₁^Δ/Δ spleen. Note the intense labeling of red pulp in both types of spleens. Fluorescent images shown in panels A-D and panels I-J were visualized with Alexa 594, mounted with Vectashield Mounting Medium with 4,6-diamidino-2-phenylindole (Vector Labs) and acquired at room temperature using a Leica DMLB fluorescence microscope (with appropriate filters) and a Spot RT Slider camera using SPOT Advanced software (Version 4.6, Diagnostic Instruments). (E-H) Visualized with diaminobenzidine and hematoxylin were taken at room temperature using an Olympus BH-2 microscope outfitted with a Nikon Coolpix 995 digital camera. Magnification bars are inserted. Some adjustments for brightness, contrast, and color balance using Adobe Photoshop were made.