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.
D015100:3,3'-Diaminobenzidine; D000085:Acetates; D019342:Acetic Acid; D000818:Animals; D001923:Brain Chemistry; D003035:Cobalt; D003346:Corticotropin-Releasing Hormone / Q000032:analysis*; D005404:Fixatives; D005557:Formaldehyde; D005949:Glucose Oxidase / Q000378:metabolism; D006651:Histocytochemistry / Q000379:methods*; D008297:Male; D010851:Picrates; D051381:Rats; D011919:Rats, Inbred Strains
D000085:Acetates; D005404:Fixatives; D010851:Picrates; D005557:Formaldehyde; D015100:3,3'-Diaminobenzidine; C026239:Bouin's solution; D003035:Cobalt; D003346:Corticotropin-Releasing Hormone; D005949:Glucose Oxidase; D019342:Acetic Acid