Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy

The main goal of this study was to develop a better light microscopic proce dure for quantitative study of the cellular co-localization of neuropeptide s in adult human brain tissue. To reach this goal, we opted for a method (p roved to be optimal on rat brain) in which sections were double immunolabel ed with two different fluorophore-conjugated secondary antibodies and analy zed with a confocal laser scanning fluorescence microscope. One of our main problems faced was a strong autofluorescence of the sections, mainly cause d by lipofuscin granules normally present in adult human brain tissue, whic h made any analysis of specific fluorescence impossible. This problem could be solved by staining the sections after immunolabeling with the dye Sudan Black B, which completely blocked this autofluorescence. The complete opti mized procedure that we eventually developed can be summarized as follows. After a relatively short fixation time (6-14 days) in 4% freshly depolymeri zed paraformaldehyde, the resected brain tissue can best be stored in a 30% sucrose solution supplemented with 0.05% NaN3 at 4C. Stored under these co nditions, cryosections from the tissue still reveal good histology and allo w successful immunocytochemical staining after a period of 6 months. Double immunolabeling is done by incubating cryo- or paraffin sections in a mixtu re of two primary antibodies directed against the targeted antigens, follow ed by incubation with two different fluorophore-conjugated secondary antibo dies. Amplification with a biotinylated secondary antibody followed by fluo rophore-conjugated streptavidin is possible. Finally, the sections are stai ned with Sudan Black B, mounted in plain 80% Tris-buffered glycerol, and st udied by confocal laser scanning fluorescence microscopy. Sections processe d in this way are well suited for qualitative and quantitative analyses of co-localized neuropeptides in human brain tissue.