Mzfliii

After dissection and fixative perfusion, brains were placed onto a sagittal brain matrix and a 2 mm-slice was cut from the middle line using razor blades. This slice was placed in a 1X PBS solution containing 10 ng/mL of Hoechst for 50 min. Thereafter, the slice was washed in a 1X PBS solution and put either on a dish for macroscopic imaging (Leica MZFLIII) or in a glass-bottom dish for confocal imaging (Leica SP8).
Serial images taken on the fluorescent macroscope were stitched with MosaicJ on Fiji to allow for the reconstruction of the whole brain. Thereafter, red channel corresponding to the mKate2 fluorescence, and bright-light channel were merged with Fiji to allow for the detection of tumor infiltrated structures.

E10.5 and E11.5 Unicorn and littermate control embryos were processed and imaged via pseudo-SEM protocol (n = 3/genotype, E10.5; n = 4/genotype, E11.5). The embryos were fixed overnight in 4% paraformaldehyde, and stained with 0.01% ethidium bromide. To ensure consistency of embryo position for photography, we photographed them in a Sylgard-coated dish with molded wells for embryo placement. We visualized the ethidium bromide signal using the DsRED filter on a Leica MZFLIII. After photography, we placed twelve landmarks on the images, and used the measure tool in Image J version 1.51w to measure the width of the face. We measured the width of several inferior and superior regions as well as the width of the singular maxillary prominence. We normalized each facial width measurement to the singular maxillary prominence width to account for subtle staging differences between individuals. The Unicorn and control embryos normalized facial widths were compared using a paired Students t-test (p < 0.05).

LRs were photographed digitally under a stereomicroscope (Leica MZ FLIII) at 60× magnification. GFP (green) and FM4-64 (red) fluorescence were observed using an LSM700 confocal laser scanning microscope (Carl Zeiss) using 488/490–555 and 555/640 nm excitation/emission filter sets, respectively. To determine the localization of the PIN : GFP fusion protein, 9-day-old seedlings were treated with FM4-64 and then incubated in half-strength liquid MS medium. The PIN8:GFP signal was quantified using the histogram function of Adobe Photoshop (Adobe Systems), as described previously (Won et al., 2009 (link)). For PIN8:GFP observation in the plasmolyzed epidermal cells of the cotyledon, 3-day-old WT or ProPIN8:PIN8:GFP transformant (in pin8) seedlings were treated with 0.05% Tween 20 for 2 h and then with 1 M mannitol for 5 to 10 min.