Optimizing Multiple Immunostaining of Neural Tissue

Interference from autofluorescence is one of the major problems in immunofluorescence analysis of neural tissue. This difficulty is experienced even with the use of laser-scanning microscopy, which can eliminate out of focus light but not the undesirable tissue-specific autofluorescence that is in focus. Therefore, we will center our discussion on different strategies to minimize neural tissue autofluorescence. The two greatest sources of autofluorescence discussed here are those induced by aldehyde fixation and by the fluorescent pigment lipofuscin, which accumulates with age in the cytoplasm of cells of the central nervous system. Finally, it is important to determine whether any treatments used to minimize autofluorescence might quench specific fluorescent compounds used to label the proteins of interest. We present the results of multi-label fluorescence immunostaining of cryostat sections of 20 μm of the human inferior colliculus. We conclude that the use of chemical agents to reduce autofluorescence and quench lipofuscin-specific fluorescence helps to maximize the fluorescent signal-to-noise ratio in immunocytochemical studies of fixed neural tissues.