A silver-staining procedure for enhancing the sensitivity of protein detection on nitrocellulose membranes in immunoblotting is described. After completing any peroxidase-Ni-diaminobenzidine immunostaining, nitrocellulose sheets are placed in a physical developer, containing sodium tungstate and ascorbic acid, until the desired grade of silver-intensification has been reached. In this way a 16- to 64-fold amplification of intensity of the initial immunostaining can be achieved. False positive silver staining of protein bands and of background are suppressed by replacing bovine serum albumin, the blocking agent most frequently used in immunoblotting, with skimmed milk.

3,3'-Diaminobenzidine↗

PubMedDate not supplied

Neurotrophic effect of hematopoietic cytokines on cholinergic and other neurons in vitro.

We examined the effects of interleukin-3 (IL-3) and other hematopoietic cytokines on the neurotransmitters, neurite formation, and differentiation in cholinergic and other types of neurons. IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), macrophage colony-stimulating factor, granulocyte colony-stimulating factor and erythropoietin (Epo) elevated choline acetyltransferase (ChAT) activity in septal cholinergic cell line SN6 as well as in primary cultured septal neurons without increasing protein contents of the cells. These effects were dose-dependent and the optimal doses were not different from those for blood cells. IL-3 had neurite-promoting activity but GM-CSF had no such effect. Both IL-3 and GM-CSF decreased intracellular acetylcholine concentration, and elevated glutamic acid decarboxylase and intracellular GABA in septal neuronal cultures. Epo elevated monoamines in PC12 cells. These effects are thought to result from direct action through their specific receptors in neurons, because (i) anti-IL-3-receptor antibody abolished the ChAT activity in septal neurons increased by IL-3; (ii) mRNA and immunoreactivity for beta subunits of IL-3 receptors were expressed in septal cholinergic neurons and (iii) presence of receptors for GM-CSF and Epo in neurons has been reported. Our observation and others strongly support that neural-immune interactions are important not only in the defense mechanism in the nervous system but also in the development, differentiation and function of neurons.

Animals↗

PubMedDate not supplied

Elastic elements in the media and adventitia of human intracranial extracerebral arteries.

We find that the media and adventitia of adult human cerebral arteries contain elastic fibers forming a dense, coherent network, similar to that found in muscular arteries of the same size in other organs. The external elastic layer in the adult human is masked for the currently employed staining methods. By treatment with 90% formic acid before fixation, the original staining character of elastic tissue can be restored. The light microscopic and scanning electron microscopic features of this network of elastic fibers are presented.

Adolescent↗