Differential subcellular localization of tubulin and the microtubule- associated protein MAP2 in brain tissue as revealed by immunocytochemistry with monoclonal hybridoma antibodies

The distribution and subcellular localization of tubulin and MAP2 in brain tissue were analyzed by immunocytochemistry with monoclonal hybridoma antibodies prepared against Chinese hamster brain tubulin and MAP2. We examined three anti-tubulin hybridoma antibodies (Tu3B, Tu9B, Tu12) specific for beta-tubulin, and two anti-MAP2 hybridoma antibodies (AP9,AP13). The specificity of each of the monoclonal antibodies was characterized by staining nitrocellulose electrophoretic blots of SDS- polyacrylamide gels of whole brain or hippocampal extracts. Each hybridoma antibody bound only its respective antigen in these preparations. Polyclonal antisera against tubulin were also examined. Sections reacted with antisera against tubulin or monoclonal antibodies against beta-tubulin revealed a wide variety of stained cellular compartments. The reaction product was found to decorate dendritic and axonal microtubles in neurons; glial cells were also stained. MAP2 immunoreactivity was found only in neurons. In the case of one of the monoclonal antibodies (AP9), staining was preferentially associated with dendritic processes. However, light but significant staining of axonal processes was seen with AP13. Within dendrites, MAP2 was found associated with dendritic microtubules and postsynaptic densities (psd), both in shaft and spine synapses. In addition, strong immunoreactivity for MAP2 was found within the cytoplasm of dendritic spines. There was little or no immunoreactivity for tubulin in the spine cytoplasm, although the psd was stained. The localization of MAP2 in dendritic spines and in the psd suggests that this protein may have a biological role independent of its association with microtubules. The observations on differential staining of the hybridoma antibodies against MAP2 suggest that there may be distinct subtypes or states of MAP2 within neurons.

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