Fig. 5
Deletion of Vhlh in liver leukocytes. a By immunohistochemistry, GFP is prominent in the collecting ducts in kidney of the reporter strain HOXB7-GFP-Cre; Vhlhfl/fl [25, 29]. b, c GFP is also detectable in single cells interspersed within the liver parenchyma (b1) or within hemangiomas (c2, 3). d Isolation of leukocytes from livers of HOXB7-Cre; Vhlhfl/fl mice. Liver cell suspensions were prepared by mincing livers, digesting with collagenase, and lysing red blood cells with red blood cell lysis buffer. Liver cell suspensions were then stained with the pan-leukocyte marker CD45 and propidium iodide (live/dead stain). Live CD45+ cells were isolated with FACS; debris and doublets were gated out. After the sort, CD45+ fractions were >90 % pure. e By PCR, Vhlh inactivation was detected in CD45+ leukocyte fraction of 5 independent samples (1–5). Samples 3–5 were additionally examined for the presence of floxed alleles (the amount of DNA samples 1 and 2 were insufficient for additional PCR tests). As control, PCR was performed without template (no template control) or with genomic DNA from a HOXB7-Cre; Vhlhfl/fl knockout kidney (positive control, which contains both Vhlh-inactivated and wild-type cells)