Eclipse c1

Mice were transcardially perfused using 4% paraformaldehyde (PFA) prepared in phosphate buffered saline (PBS) and tissues harvested were postfixed in 4% PFA overnight. Brains were sagittally sectioned to 50um thick sections using vibratome (Leica VT 1000S) and stored in a solution of 1xPBS with 0.05% sodium azide. Similarly lungs, testis, and oviducts were sectioned coronally. For immunohistochemical analysis tissue sections were immersed in blocking buffer (10% goat serum and 1% Triton-X-100 in 1xPBS) for an hour at room temperature (RT). Following blocking, sections were incubated with appropriate primary antibodies [chicken anti-GFP (Abcam) at 1:2000; rabbit anti-RFP (Abcam) at 1:1000; mouse anti-Tubulin (Sigma) at 1:1000; rabbit S100 (DAKO) at 1:1000] in antibody diluent (1% goat serum and 0.03% Triton-X-100 in 1xPBS) for overnight at 4°C. Sections were then washed three times, 5 minutes each, using 1xPBS and incubated with appropriate secondary antibodies in antibody diluent with DAPI [(Sigma) at 1µg/ mL] for an hour at RT. Following PBS washes, sections were mounted to plus-sided slides and coverslip placed on sections with mounting media to avoid air bubbles. Confocal images of stained sections were captured using either Nikon Eclipse C1 or Olympus fluoview FV1000 confocal microscopes.