Temperature, peroxide concentration, and immunohistochemical staining method affects staining intensity,...

The techniques used to label neural tissue for specific antigens can vary significantly. Some immunostaining methods use free-floating tissue sections, whereas others use tissue sections mounted on slides. Mounting sections on glass slides before labeling the tissue with antigens is preferred method for neonatal tissue; processing young tissue by free-floating methods often destroy it. Surprisingly optimal temperature for storing tissue can vary with age. This study describes parameters developed to obtain robust staining of both young and old tissue. Our results show the most robust staining was found in tissue that was (1) stored at very low temperatures (-20 degrees C and -80 degrees C), (2) pretreated with 0.01% peroxide, and (3) entirely immersed in the staining solutions during immunohistochemistry.