Figure 3. Characterization of Ccl19-Cre activity and DL1 expression in stromal cells of the spleen. (A) Spleen sections of R26-EYFP (control) and R26-EYFPΔCcl19 mice stained for EYFP and collagen IV expression. EYFP staining is indicative for Cre recombinase activity through the deletion of the lox-stop-lox cassette in the R26 gene locus. Collagen IV highlights the basement membrane of blood vessels and the marginal sinus. The dashed box is represented as a higher magnification on the right side. B, B cell follicle; T, T cell zone. Arrow head pointing to EYFP reticular cells. FDCs; MRC, marginal fibroblastic reticular cells; BRCs; FRC, T zone fibroblastic reticular cells; CA, central arteriole. (B) Representative FACS profile of Tomato+ (Ccl19-Cre−) and GFP+(Ccl19-Cre+) splenic stromal cells isolated from mTmGΔ/+Ccl19 reporter mice gated on Aqua−Ter119−CD45−CD35− cells and further analyzed for the expression of podoplanin (Pdpn, gp38+) and CD31. GFP+ cells represent the fraction of cells that express Ccl19-Cre. Percentages indicate the mean frequency among CD35− stromal cells (n = 5). (C and D) Cell populations from digested spleens of three individual mTmGΔ/+Ccl19 mice were sorted as indicated in B, pooled, and assessed by semiquantitative PCR for the deleted (Δ) versus undeleted (loxP) DL1 gene (C) and by qRT-PCR for relative mRNA expression levels (D). The numbers in D indicate the fold enrichment relative to total WT splenocytes. Bars in D represent data from triplicate PCR reactions normalized to the HPRT housekeeping gene of pooled sorted stromal cells from 3 individual mice for the indicated reporter line. (E) Splenic sections of DL1lox/lox and DL1Δ/ΔCcl19 mice were stained for DL1 (top), Pdpn, and desmin (bottom) as indicated. Arrows point to different sites of DL1 expression and stars denote examples of background staining (in form of larger dots) due to endogenous horseradish peroxidase activity. The dashed boxes are represented as a higher magnifications shown below the images. Experiments shown in A, B, and E are representative of two to five experiments with at least three different mice per group.
Figure 3.

Characterization of Ccl19-Cre activity and DL1 expression in stromal cells of the spleen. (A) Spleen sections of R26-EYFP (control) and R26-EYFPΔCcl19 mice stained for EYFP and collagen IV expression. EYFP staining is indicative for Cre recombinase activity through the deletion of the lox-stop-lox cassette in the R26 gene locus. Collagen IV highlights the basement membrane of blood vessels and the marginal sinus. The dashed box is represented as a higher magnification on the right side. B, B cell follicle; T, T cell zone. Arrow head pointing to EYFP reticular cells. FDCs; MRC, marginal fibroblastic reticular cells; BRCs; FRC, T zone fibroblastic reticular cells; CA, central arteriole. (B) Representative FACS profile of Tomato+ (Ccl19-Cre−) and GFP+(Ccl19-Cre+) splenic stromal cells isolated from mTmGΔ/+Ccl19 reporter mice gated on Aqua−Ter119−CD45−CD35− cells and further analyzed for the expression of podoplanin (Pdpn, gp38+) and CD31. GFP+ cells represent the fraction of cells that express Ccl19-Cre. Percentages indicate the mean frequency among CD35− stromal cells (n = 5). (C and D) Cell populations from digested spleens of three individual mTmGΔ/+Ccl19 mice were sorted as indicated in B, pooled, and assessed by semiquantitative PCR for the deleted (Δ) versus undeleted (loxP) DL1 gene (C) and by qRT-PCR for relative mRNA expression levels (D). The numbers in D indicate the fold enrichment relative to total WT splenocytes. Bars in D represent data from triplicate PCR reactions normalized to the HPRT housekeeping gene of pooled sorted stromal cells from 3 individual mice for the indicated reporter line. (E) Splenic sections of DL1lox/lox and DL1Δ/ΔCcl19 mice were stained for DL1 (top), Pdpn, and desmin (bottom) as indicated. Arrows point to different sites of DL1 expression and stars denote examples of background staining (in form of larger dots) due to endogenous horseradish peroxidase activity. The dashed boxes are represented as a higher magnifications shown below the images. Experiments shown in A, B, and E are representative of two to five experiments with at least three different mice per group.

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