(A) Schematic of the Cre-dependent reporter present in the Ai14D strain. Reporter cassette, inserted at the safe harbor Rosa26 locus, carries a strong pCAG promoter followed by a LoxP-flanked STOP cassette with three repeated in frame sequences. In the absence of Cre activity, transcription through this construct leads to transcriptional termination and no reporter activity. Following Cre-dependent excision of the LoxP cassette, the pCAG promoter is juxtaposed adjacent to a sequence encoding a tandem Tomato (tdTomato) fluorescent protein, resulting in robust expression of Tomato in tissues expressing Cre. (B) Lightsheet imaging of the mouse epididymis in the negative control Ai14D background. Following animal sacrifice, tissues were cleared for imaging according to a modified CLARITY protocol (Materials and methods). The entire epididymis, along with a large section of the vas deferens, was then imaged using the LaVision Biotec lightsheet microscope. Regions corresponding to the proximal (caput), middle (corpus), and distal (cauda) epididymis, and the vas deferens, are indicated on the image of the entire tissue sample. Insets show representative 2D slices of the indicated anatomical regions. (C) Positive control showing robust Tomato expression driven by the caput-specific Defb41 Cre driver (Björkgren et al., 2012). Panels show whole tissue image and representative sections as described in panel (B), along with representative sections of the kidney and liver, as indicated. See also Figure 1—figure supplement 1.