Confirmation of high recombination efficiency in R26 ^Flpe ;Col4a1 ^+/Tag1 mice. (A) qPCR analyses showing that the expression levels of Col4a1 in lungs from 6-mo-old R26^Flpe;Col4a1^+/Tag1 mice were reduced to ∼50% of those from control R26^Flpe;Col4a1^+/+ mice. The expression levels of Col4a2 were not affected (P = 0.162). n = 6 for each genotype. (B) Representative western blot images (left) and quantification (right) showing that COL4A1 protein levels in lung lysates from 6-mo-old R26^Flpe;Col4a1^+/Tag1 mice were significantly reduced compared with control R26^Flpe;Col4a1^+/+ mice. COL4A2 protein levels were not affected (P = 0.397). n = 5 and 7 for R26^Flpe;Col4a1^+/+ and R26^Flpe;Col4a1^+/Tag1 mice, respectively. Data are presented as mean ± SEM. **P < 0.01; ****P < 0.0001; NS, not significant, unpaired Student’s t test. (C) Representative immunofluorescence images of brain sections from P7 mice labeled with anti-COLIV (green) and anti-mCherry (magenta) antibodies. Sections were counter-stained with DAPI (blue). In Col4a1^+/Tag1 mice in the absence of Flp, the mCherry tagged COL4A1 isoforms localized to the BMs of blood vessels as indicated by co-labeling with anti-COLIV. In R26^Flpe;Col4a1^+/Tag1 mice, nearly all of the mCherry signal was eliminated from the BMs indicating successful Flp-dependent recombination of the tagged allele. The remaining mCherry signal labels cells actively producing COL4A1 or rare instances of incomplete recombination. Scale bar = 100 μm. (D) Magnified images of boxed areas in A. Arrows indicate colocalization of mCherry and COLIV signals. Arrowheads indicate intracellular mCherry signals, and asterisks indicate the absence of mCherry signals from the blood vessel. n = 3 per genotype from three litters. Scale bar = 20 μm. Source data are available for this figure: SourceData FS2.