Figure 3
Figure 3. Immunostaining of FIX in 2bF9 mice. (A-F) Confocal microscopy study of FIX in platelets. Platelets isolated from 2bF9-H (A-C) and FIXnull mice (D-F) were immunofluorescently stained for FIX (green) and VWF (red). The merged images (C,F) show that FIX was expressed in 2bF9 transgenic platelets and colocalized with VWF. Bars represent 15 μm. (G-L) Confocal microscopy study of FIX in mononuclear cells. Mononuclear cells and platelets isolated from 2bF9-H (G-I) or FIXnull (J-L) mice were stained for FIX (green). FIX was detected in 2bF9-H platelets, but no FIX was detected in mononuclear cells. Bars represent 10 μm. (M-P) Immunoelectron microscopy study of FIX in platelets. Platelets from 2bF9-H (M-N) and FIXnull mice (O-P) were immunogold stained for FIX (5-nm colloidal gold, indicated by arrows) and VWF (10-nm colloidal gold, indicated by arrowheads). FIX colocalized with VWF in α-granules. Bars represent 100 nm. (Q-T) Femoral marrow from 2bF9-H (Q,S-T) and FIXnull mice (R) were immunostained for FIX (Q-R) or with an isotype control (S). 2bF9-H marrow was also stained with hematoxylin and eosin (T) to demonstrate the morphology of megakaryocytes. FIX protein was demonstrated in megakaryocytes of 2bF9-H mice. Arrows point to representative megakaryocytes in each figure. Bars represent 50 μm. These results demonstrate that FIX was expressed in platelet lineage cells and stored in the platelet α-granules of 2bF9 transgenic mice. Confocal images (A-L) were acquired using an Olympus FV-1000-MPE Multiphoton Confocal Microscope (Olympus Corporation) with a 100×/1.4 NA oil objective with 2×digital zoom into Imaging Software FV10-ASW Version 2.00a (Flowview Software) using Vectashield Mounting Medium (Vector Labs). Cofocal specimens were mounted using Vectashield Mounting Medium (Vector Labs). EM images (M-P) were acquired using a JEOL JEM2100 Electron Microscope (JEOL USA Inc.) at 100 000× magnification with a Gatan Ultrascan 1000 CCD Camera (Gatan Inc) into Digital Micrograph Software Version GMS 1.8.4 (Gatan Inc). Immunohistochemistry images (Q-T) were acquired using a Nikon Eclipse E600 Microscope (Nikon) with a 40×/0.75 NA objective using a SPOT Insight Firewire Color Mosaic Model 11.2 Digital Camera (SPOT Insight Solutions) into SPOT Software Version 4.1. Immunohistochemistry specimens were mounted using Mounting Medium (Pierce).

Immunostaining of FIX in 2bF9 mice. (A-F) Confocal microscopy study of FIX in platelets. Platelets isolated from 2bF9-H (A-C) and FIXnull mice (D-F) were immunofluorescently stained for FIX (green) and VWF (red). The merged images (C,F) show that FIX was expressed in 2bF9 transgenic platelets and colocalized with VWF. Bars represent 15 μm. (G-L) Confocal microscopy study of FIX in mononuclear cells. Mononuclear cells and platelets isolated from 2bF9-H (G-I) or FIXnull (J-L) mice were stained for FIX (green). FIX was detected in 2bF9-H platelets, but no FIX was detected in mononuclear cells. Bars represent 10 μm. (M-P) Immunoelectron microscopy study of FIX in platelets. Platelets from 2bF9-H (M-N) and FIXnull mice (O-P) were immunogold stained for FIX (5-nm colloidal gold, indicated by arrows) and VWF (10-nm colloidal gold, indicated by arrowheads). FIX colocalized with VWF in α-granules. Bars represent 100 nm. (Q-T) Femoral marrow from 2bF9-H (Q,S-T) and FIXnull mice (R) were immunostained for FIX (Q-R) or with an isotype control (S). 2bF9-H marrow was also stained with hematoxylin and eosin (T) to demonstrate the morphology of megakaryocytes. FIX protein was demonstrated in megakaryocytes of 2bF9-H mice. Arrows point to representative megakaryocytes in each figure. Bars represent 50 μm. These results demonstrate that FIX was expressed in platelet lineage cells and stored in the platelet α-granules of 2bF9 transgenic mice. Confocal images (A-L) were acquired using an Olympus FV-1000-MPE Multiphoton Confocal Microscope (Olympus Corporation) with a 100×/1.4 NA oil objective with 2×digital zoom into Imaging Software FV10-ASW Version 2.00a (Flowview Software) using Vectashield Mounting Medium (Vector Labs). Cofocal specimens were mounted using Vectashield Mounting Medium (Vector Labs). EM images (M-P) were acquired using a JEOL JEM2100 Electron Microscope (JEOL USA Inc.) at 100 000× magnification with a Gatan Ultrascan 1000 CCD Camera (Gatan Inc) into Digital Micrograph Software Version GMS 1.8.4 (Gatan Inc). Immunohistochemistry images (Q-T) were acquired using a Nikon Eclipse E600 Microscope (Nikon) with a 40×/0.75 NA objective using a SPOT Insight Firewire Color Mosaic Model 11.2 Digital Camera (SPOT Insight Solutions) into SPOT Software Version 4.1. Immunohistochemistry specimens were mounted using Mounting Medium (Pierce).

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