Nonspecific labeling of myelin with secondary antisera and high concentrations of triton X-100

Abstract

Triton X-100 is used in immunohistochemistry to make tissue permeable, to present certain antigens to antisera, and to prevent certain nonspecific interactions. This detergent is routinely dissolved in buffers at concentrations of 0.01-0.2%. Using high concentrations of Triton X-100 (0.2- 2%) and anti-immunoglobulins G (anti-IgGs), labeling of myelin and microglia was detected in fixed brain tissue by indirect fluorescence and avidinbiotin- immunoperoxidase techniques. Differences were found between the species studied (mouse and rat), the type of anti-IgG (anti-mouse, anti-rabbit, anti- sheep, anti-rat, or anti-guinea pig), the detergent concentration, and whether Triton X-100 was included in the incubation media or applied as a pretreatment. Mouse brain displayed strong myelin labeling with all anti- IgGs but rat brain only with anti-rabbit or anti-sheep IgGs. Staining of ramified microglia occurred only in mouse tissue when anti-mouse IgG was used. Nonspecific staining of myelin was also intense in paraffin-embedded tissue and in human brain frozen sections. These results are significant for the prevention of undesirable staining in routine immunolabeling and they also provide a comparatively inexpensive, easy to perform strong labeling of myelin. In addition, the double marker signal (peroxidase and fluorescence) is useful for double labeling studies.