BABB vs Prolong Gold analysis

Two pools of embryos were stained with the Crumbs antibody (1:10, DSHB Cq4 supernatant) and an Alexa Fluor 488 (1:500, Invitrogen) in the microplate. One pool was mounted on the multiwell slide in Prolong Gold and the other eluted into ethanol, cleared in BABB, and mounted on the multiwell slide. Ten embryos were imaged for each condition.

In Fiji, stacks were resliced using the Reslice[/] command to view the embryos along the x–z axis. Resliced images were projected using the Z Project command for the Max Intensity.

To create the composite representations, each projection was concatenated together using the Concatenate command and projected again with Z Project for the Average Intensity. Brightness and contrast were enhanced for clarity. Because BABB and Prolong Gold have different indexes of refraction²², composite images were stretched based on the following formulas previously derived^23,24:

To create the plot in Fig. ​Fig.4B,4B, the concatenated stacks were rotated − 90° using the Rotate command. A box was drawn through the center of the embryo using the “Rectangle” selection tool. Intensities within this region of interest were measured using the Plot Profile function. Data were normalized in both intensity and embryo length and plotted using MATLAB.