R t u vectastain goat anti rabbit mouse kit

For histological examination, X-gal + glands were embedded in parafin and cut in 5 μm sections and mounted on positively charged slides. Sections from samples were counter stained with nuclear fast red to identify lacZ+ structures. Sections were subsequently cleared in xylenes and rehydrated through ethanol gradients. Antigen retrieval was performed by heating slides in a boiling water bath for 20 min. in Tris-EDTA pH 9.0 (Dako). Using 3% hydrogen peroxide for 15 minutes at room temperature removed endogenous peroxidase activity. Slides were blocked with normal horse serum (Vector Laboratories, Burlingame, CA) for 1 hour at room temperature and then incubated overnight with primary antibodies to smooth muscle actin (Zymed) or progesterone receptor (Dako) at 4°C. Secondary antibody staining was performed using the R.T.U. Vectastain (goat anti-rabbit/mouse) kit (Vector Laboratories). Staining was visualized using the DAB peroxidase substrate kit (Vector Laboratories) per manufacturer's recommendations. Slides were counterstained with Mayer's hematoxylin (Sigma-Aldrich) and negative tissue controls were included in all immunohistochemical analyses.