Immunodetection of prostaglandin D synthase: conditions of localization in a defined subclass of primary sensory neurons.

Prostaglandin (PG) D2 is synthesized by primary sensory neurons grown in vitro. The question can be raised of whether the entire population or only a particular subpopulation of primary sensory neurons synthesizes PGD2 in vivo. To clarify this issue it was necessary to demonstrate that PGD synthase activity persists in fresh dorsal root ganglion (DRG) cryostat slices by characterizing newly formed PGD2 from [14C]-arachidonic acid, and to determine by immunocytochemistry and to identify at the ultrastructural level the neuron subpopulation expressing glutathione (GSH)-independent PGD synthase. Among the various procedures tested, the most intense, selective, and reproducible immunostaining pattern was obtained after periodate-lysine-formaldehyde fixation in phosphate buffer, permeabilization with 0.25% Triton X-100, and incubation with 10 micrograms/ml purified antibodies. Under these conditions, a subpopulation of small Class B ganglion cells was strongly immunoreactive, whereas adjacent control sections treated with absorbed antibodies or with non-immune rabbit or goat serum were unreactive. To identify the subclass of the immunoreactive small Class B neurons, immunostained vibratome slices of DRG were embedded in Epon. Ganglion cell bodies loaded with immunoprecipitates in superficially cut sections were first identified and then ultrastructurally analyzed in thin sections taken from a deeper level to obtain improved preservation of the cell architecture. This procedure enabled us to demonstrate that GSH-independent PGD synthase is accumulated in Subclass B1 primary sensory neurons.