Figure 2. Immunohistochemical analysis of MCF-7 and SOX2-overexpressing MCF-7/Rep xenotumors. Top: H&E, ERα, and PR immunohistochemical staining in MCF-7 and SOX2-overexpressing MCF-7/Rep (clone #3) xenotumor tissues. Bottom: the Masson trichrome stain was used to distinguish collagen from smooth muscle and to identify an increase in collagenous tissue in xenograft specimens (muscle fibers-red, collagen-blue, fibrin-pink, cytoplasm-red, and nuclei-blue/black). The combination of PAS and PAS with diastase (PASD) was used for demonstration of glycogen content. PAS stains magenta the glycogen stores, but it also stains with the same color additional tissue elements (neutral mucosubstances, epithelial sulfomucins, and sialomucins). PASD reaction digests glycogen with a pretreatment of the tissue with α-amylase (depolymerizes glycogen), and as a result of this, any magenta staining is solely attributed to the other tissue elements described above but not to glycogen. The Alcian Blue stain was used for the demonstration of neutral and acidic mucosubstances. Alcian Blue imparts a blue color to the acidic mucins and other carboxylated or weakly sulphated acid mucosubstances. Because the PAS reaction stains basement membranes, glycogen, and neutral mucosubstances pink to red, mixtures of neutral and acidic mucosubstances will appear purple due to positive reactions with both Alcian Blue and PAS. Original magnification, ×200.