Optimized Whole-Mount X-gal Staining to Detect Hedgehog Signaling Activity in Basal Cell Carcinoma: A Platform for Spatial Integration with Immune Analysis.

Whole-mount X-gal staining is a classical histochemical method for detecting β-galactosidase (LacZ) expression in fixed tissues, providing spatial resolution of gene activity in situ. In cancer research, LacZ serves as a versatile reporter for monitoring gene activation, tracing cell lineage in genetically engineered models, and assessing cellular responses to oncogenic signaling within the tumor microenvironment. Here, we present an optimized protocol for the in situ visualization of LacZ+ cells in the skin of Ptch1+/- SKH-1 mice-a genetically defined model of basal cell carcinoma (BCC) characterized by constitutive Hedgehog (Hh) pathway activation. In this model, LacZ expression faithfully reports Hh signaling and allows direct visualization of emerging and established BCC lesions. Importantly, our protocol preserves tissue integrity and antigenicity, enabling seamless integration with downstream immunostaining or multispectral immunofluorescence. When combined with immune markers-including those for regulatory T cells, cytotoxic T lymphocytes, myeloid subsets, and cytokine expression-this approach permits high-resolution spatial profiling of immune architecture in relation to LacZ+ tumor foci. This method is particularly suited for studying how oncogenic signaling pathways such as Hh shape the immune landscape during tumor initiation, progression, or therapeutic response. Overall, the protocol offers a versatile platform for coupling gene expression mapping with immune contexture analysis in preclinical models of skin cancer.