The ability to detect transient changes in molecular networks lies at the heart of cancer biology research. This is especially apparent during tumorigenesis, where initiating mutations typically affect mitogens and cell-cycle molecules such as PDGF or retinoblastoma protein (Rb). One of the primary consequences of such processes is the inappropriate stimulation of downstream targets, normally through posttranslational modification. Immunohistochemistry (IHC) provides an important tool for assessing such changes in situ, permitting different aspects of tumor biology to be examined as a tissue undergoes transformation. Nevertheless, this can be difficult to achieve, particularly in complex environments like the brain. Here, we provide the automated methodology we have employed for the successful detection of phosphorylation of S6 ribosomal protein (S6-RP) and the retinoblastoma protein (Rb) in response to PDGF stimulation in a mouse model of glial brain tumor development.