488 nm conjugated donkey anti mouse igg

The lower body of postnatal day 0 (P0) or E11.5 mice were fixed overnight in 4% paraformaldehyde, paraffin-embedded, and sectioned at 6 μm for hematoxylin and eosin (H&E) staining or immunohistochemistry. For immunohistochemistry, tissues were deparaffinized and rehydrated. Antigen retrieval was performed by boiling the sections in 0.1 M sodium citrate buffer for 30 minutes. Samples were blocked for 30 minutes in PBST with 10% FBS. Sections were incubated with primary antibody overnight at 4°C. The primary antibodies used were mouse monoclonal anti-E-cadherin (BD Biosciences, 1:100, Franklin Lakes, NJ), mouse monoclonal calbindin-D-28K antibody (Sigma-Aldrich, 1:100), rabbit polyclonal cleaved caspase-3 antibody (Cell Signaling Technology, 1:100), and/or mouse monoclonal alpha smooth muscle actin antibody (Thermo Fisher Scientific, 1:100). Sections were washed with PBS and incubated at 4°C overnight with secondary antibodies, 488 nm conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:250) and/or 594 nm conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, 1:250). Sections were visualized using a Leica DM 2500 light and fluorescent microscope and photographed with Leica DFC7000T camera, using Leica Application Suite X software.

Embryos at E11.5 were dissected and urogenital ridges ventral to the hindlimb were exposed. Tissues were dehydrated through a methanol series and stored at −20°C. Tissues were rehydrated through a methanol series and washed with phosphate-buffered saline (PBS) with 0.1% Tween20 (PBST) three times for 10 minutes. Tissues were blocked with 10% fetal bovine serum (FBS) in PBST for one hour. Samples were incubated with primary antibody at 4°C overnight. Primary antibodies used were mouse monoclonal anti-Calbindin-D-28K (Sigma-Aldrich, 1:100, St. Louis, MO) and rabbit polyclonal anti-cleaved Caspase-3 (Cell Signaling Technology, 1:250, Danvers, MA). The tissues were washed with PBST five times, for one hour each, then incubated overnight at 4°C with secondary antibodies, including 488 nm conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, 1:250, West Grove, PA) and 594 nm conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, 1:250). Tissues were washed with PBST five times, for one hour, and visualized under Leica M165FC Stereo Microscope and photographed with a QImaging Qiacam Fast 1394 camera, using QCapture software (QImaging, Surrey, BC, Canada). Common nephric duct length (caudal end of the Wolffian duct at the cloaca to the cranial edge of the ureteric bud stalk) was measured using ImageJ software (National Institutes of Health, Bethesda, MD) [27 (link)].