Double immunocytochemistry in pre-embedding electron microscopy for the detection of neurotensin and tyrosine hydroxylase in the guinea pig, using two primary antisera raised in the same species.

In this study we identified for electron microscopy two different antigens (neurotensin and tyrosine hydroxylase) in the same pre-embedding section of nervous tissue, using two antibodies obtained in the same species. Optimal ultrastructural results were obtained without adding to the fixative either glutaraldehyde or acrolein (normally used for electron microscopy techniques). The different developing methods used in this study (DAB in combination with either 1 nm silver-enhanced colloidal gold or benzidine dihydrochloride) are perfectly distinguishable at the ultrastructural level, and show some advantages over other previously described developing procedures. For instance, the use of small gold particles (1 nm) reduces the severity of membrane damage caused by tissue penetration of the bigger gold particles (5 nm). In addition, the reaction products are stable, so there is no need to stabilize them before osmication, as is necessary in other developing methods such as the TMB procedure. The immunolabeling results obtained in this study were similar in both developing methods, although synaptic profiles were more readily visible when the DAB/colloidal gold procedure was used. Using electron microscopy, we have detected TH immunoreactivity in dendrites and perikarya receiving synaptic contacts from NT-positive terminals, as well as TH-immunoreactive inputs on NT-positive neurons, at both the somatic and dendritic levels.