Improved fixation and cobalt-glucose oxidase-diaminobenzidine intensification for immunohistochemical demonstration of corticotropin-releasing factor in rat brain.

An optimal fixation method and intensification procedure may be required in brain immunohistochemistry to obtain intense and widespread staining for a specific antigen, in cases where ordinary fixation and conventional immunohistochemistry result in only partial demonstration of the antigen. In the present study of localization of corticotropin-releasing factor immunoreactivity (CRFI) in rat brain, the importance of such intensification is shown. We describe a fixation procedure in which perfusion of rat brain with Bouin's solution is followed by a PBS wash and a further perfusion with either Zamboni's fluid or 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4), for subsequent investigation of the detailed localization of CRFI in cerebral cortex and subcortical structures. The cobalt-glucose oxidase-diaminobenzidine (Co-GOD) intensification method has been modified to increase the sensitivity of immunostaining by reducing the concentration of glucose oxidase, which is added to the final incubation solution as a generator of hydrogen peroxide. The use of cobalt acetate instead of cobalt chloride appears to slightly suppress background staining in the Co-GOD method. Combination of the two modified procedures was applied to visualize intense and widespread CRFI in a variety of rat brain regions, including median eminence, cerebral cortex, and central amygdaloid nucleus.