Confocal microscopy
Kidneys were dissected, fixed in paraformaldehyde, treated with methanol, and washed with PBS containing Tween-20 (PBS-T) prior to treatment with lectins and antibodies. PBS-T was used for incubations of the tissues with fluoroscein-conjugated Dilichos biflorus aggutinin (DBA, Vector; Burlingame, USA), and PBS-T plus 2% goat serum was used for incubations with the antibodies. The primary antibodies were anti-WT1 (c-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-uvomorulin (e-cadherin, Cdh1, Sigma-Aldrich St. Louis, MO, USA), and anti-Cited1 (Neomarkers-Lab Vision Corporation, Fremont, CA, USA). The secondary antibodies were Alexa 555-conjugated anti-rabbit and Alexa 633-conjugated anti-rat antibodies (Molecular Probes, Eugene, OR, USA).
The tissues were imaged with a laser scanning microscope (Carl Zeiss, Thornwood, New York, USA) equipped with an argon (488 nm) and two HeNe lasers (543 nm and 633 nm). Optical sections approximately 2 μm thick were obtained every 5 μm to a depth of at least 65 μm. The sections began at the surface of the kidney and were on a plane tangential to it. Two Z-stack series were obtained, one from each of the two kidneys of each embryo. Ureteric bud tips identified by section tracing were counted within a defined area of the confocal image.