Figure 6

Demonstration of expression of the goat uromodulin – GFP transgene and endogenous uromodulin in kidney sections by immunostaining. (A). H & E kidney TAL region staining of a control non-transgenic mouse. The arrows show microstructures with thick walls indicative of the thick segment of the ascending limb of Henle's loop within the TAL region. Fixed kidney sections from a negative control mouse (B) and the 99-122-1-A5M transgenic mouse (C) were stained with a polyclonal anti-human uromodulin antibody, followed by treatment with Texas-Red conjugated secondary anti rabbit IgG antibody. Expression of endogenous uromodulin in the cells (red) appears in a punctuated pattern, whereas the GFP expression is cytoplasmic (green). Immunostaining of similar sections for uromodulin confirmed that expression is restricted to tubular epithelial cells of these structures (B, C) indicated by red. The same sections observed for GFP expression by confocal microscopy indicated that expression was restricted to similar structures as in B &C but not co-expressed with the endogenous uromodulin staining. As expected GFP was absent in the renal sections obtained from a non-transgenic mouse. Both GFP (D, F) (green) and uromodulin (E, F) (red) were co-expressed in cells in the TAL segment (transgenic mouse, 99-122-1-A5M).