Enhanced Immunohistochemical Detection of Autonomic Nerve Fibers, Cytokines and Inducible Nitric Oxide Synthase by Light and Fluorescent Microscopy in Rat Spleen

Abstract

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-βeta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor -α (TNF-α), interferon-γ (IFN-γ), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-α, iNOS, IFN-γ and c-fos, animals were injected IV with saline or 100 μg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-μm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-γ, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-α, IFN-γ, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.