Carbodiimide as a tissue fixative in histamine immunohistochemistry and its application in developmental neurobiology

The object of this study was to develop an immunohistochemical method that could be used to study neuronal histamine, especially in nerve fibers and terminals where most previous methods have not been applicable. Three new antisera were produced in rabbits against conjugated histamine, and the fixative used in conjugation, 1-ethyl-3(3-diamethylaminopropyl)-carbodiimide (EDCDI), was used in tissue fixation and compared to paraformaldehyde. Specificity of the antisera was established with dot-blot tests on nitrocellulose, with blocking controls and affinity-purified antibodies. EDCDI appeared to be superior to paraformaldehyde as a fixative, and histamine-immunoreactive nerve cells were visualized in developing rat brain during late fetal development from embryonal day 12. By the second postnatal week, the distribution of histamine-immunoreactive neurons in rat brain had reached the adult pattern and immunoreactive nerve fibers were seen in many areas. Posterior hypothalamic neurons from newborn rat in vitro showed strong immunoreactivity for histamine and developed long varicose fibers, which covered the culture dish by the end of the fourth week in vitro. Fixation with EDCDI also allowed detection of histamine in gastric enterochromaffin-like cells and mast cells in rat. The results suggest that the histamine-containing neuron system in rat brain develops during the late fetal and early postnatal periods, and that immunoreactive neurons develop long fibers both in vivo and in vitro.