Fig. 2.
Fig. 2. Localization of RAG1/GFP signal after immunization with TD-Ags in rag1/gfp knockin mice. / (A) Site of RAG1 expression in the spleen. GFP signal under confocal laser microscopy was compared to the immunohistochemical analysis of the GC area of the spleen after immunization with SRBCs. SRBCs were injected into the peritoneal cavity of heterozygous rag1/gfpknockin mice (9 weeks old). On days 0, 5, 10, and 15 after immunization, spleens were obtained and processed as described in “Materials and methods.” GFP signal (green) was examined under laser microscopy. The same section was then stained with biotinylated-PNA followed by streptavidin-HRP with DAB staining (brown). Original magnification, 100 ×. (B) Site of RAG1 expression in lymph nodes. Sizes of GCs and localization of GFP signals were compared on the same or serial sections of lymph nodes 8 or 16 days after Ag immunization. NP-CGG (20 μg) in CFA was injected into foot pads of rag1/gfp knockin mice (6 weeks old), and cryosections were prepared from popliteal lymph nodes after 8 and 16 days. First, GFP signal was analyzed using laser microscopy. Then the same section was stained with H&E, and serial sections were analyzed using biotinylated-PNA or rat anti–GL-7 mAb, respectively. Expression of PNA and GL-7 was visualized with DAB staining using streptavidin-HRP (brown). More than 50 GCs from spleens and lymph nodes were observed after immunization with TD-Ags, but none of the GCs showed positive GFP signals within the GC area when analyzed as shown in this figure. (C) Flow cytometric analysis of GFP expression in the spleen. Heterozygousrag1/gfp knockin mice (9 weeks old) and WT mice were immunized with TD-Ags, and the spleen cells were examined after surface staining for B220 and Fas. GFP signal was compared in B220+Fas+ cells before and after the immunization. (D) Flow cytometric analysis of GFP signal in the large cells after immunization (15 days). Heterozygous rag1/gfpknockin mice (9 weeks old) were untreated or immunized with TD-Ags, and splenic B220+ B cells were gated using forward scatter (FSC) to separate R1 (small cell) and R2 (large cell) populations. For comparison, the GFP signal for the RAG1/GFP splenocytes was superimposed over the contour of wild-type splenocytes.

Localization of RAG1/GFP signal after immunization with TD-Ags in rag1/gfp knockin mice.

(A) Site of RAG1 expression in the spleen. GFP signal under confocal laser microscopy was compared to the immunohistochemical analysis of the GC area of the spleen after immunization with SRBCs. SRBCs were injected into the peritoneal cavity of heterozygous rag1/gfpknockin mice (9 weeks old). On days 0, 5, 10, and 15 after immunization, spleens were obtained and processed as described in “Materials and methods.” GFP signal (green) was examined under laser microscopy. The same section was then stained with biotinylated-PNA followed by streptavidin-HRP with DAB staining (brown). Original magnification, 100 ×. (B) Site of RAG1 expression in lymph nodes. Sizes of GCs and localization of GFP signals were compared on the same or serial sections of lymph nodes 8 or 16 days after Ag immunization. NP-CGG (20 μg) in CFA was injected into foot pads of rag1/gfp knockin mice (6 weeks old), and cryosections were prepared from popliteal lymph nodes after 8 and 16 days. First, GFP signal was analyzed using laser microscopy. Then the same section was stained with H&E, and serial sections were analyzed using biotinylated-PNA or rat anti–GL-7 mAb, respectively. Expression of PNA and GL-7 was visualized with DAB staining using streptavidin-HRP (brown). More than 50 GCs from spleens and lymph nodes were observed after immunization with TD-Ags, but none of the GCs showed positive GFP signals within the GC area when analyzed as shown in this figure. (C) Flow cytometric analysis of GFP expression in the spleen. Heterozygousrag1/gfp knockin mice (9 weeks old) and WT mice were immunized with TD-Ags, and the spleen cells were examined after surface staining for B220 and Fas. GFP signal was compared in B220+Fas+ cells before and after the immunization. (D) Flow cytometric analysis of GFP signal in the large cells after immunization (15 days). Heterozygous rag1/gfpknockin mice (9 weeks old) were untreated or immunized with TD-Ags, and splenic B220+ B cells were gated using forward scatter (FSC) to separate R1 (small cell) and R2 (large cell) populations. For comparison, the GFP signal for the RAG1/GFP splenocytes was superimposed over the contour of wild-type splenocytes.

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