Jan De Mey

Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes

A sensitive staining method for protein blots on nitrocellulose membranes is described and compared with commonly used dye staining methods. It uses colloidal metal sols (gold or silver) stabilized with Tween 20 and adjusted to pH 3. It is based on the selective high-affinity binding of colloidal metal particles to the proteins and produces a red-purplish color (gold) or dark grey (silver). The sensitivity of this new staining method is in the same range as silver staining of polyacrylamide gels and matches the sensitivity of overlay assays. It will therefore be a useful tool for correlating the position of bands or spots of proteins detected with overlay assays with the complete electropherogram in a duplicate protein blot.

Electron immunocytochemical localization of enkephalin-like material in catecholamine-containing cells of the carotid body, the adrenal medulla, and in pheochromocytomas of man and other mammals.

Enkephalin-like immunoreactivity has been localized to electron-dense secretory granules of cat and piglet carotid bodies and adrenal medullae, horse adrenal medulla, and also to human adrenal medulla and pheochromocytomas using a gold-labeled antibody technique performed at the electron microscopic level. The same granules were also demonstrated to exhibit dopamine-beta-hydroxylase-like immunoreactivity, which suggests a granular colocalization of amines and peptides in catecholamine-storing cells.

Sequential use of the PAP and immunogold staining methods for the light microscopical double staining of tissue antigens: Its application to the study of regulatory peptides in the gut

Abstract Double immunoperoxidase staining using different couplers can give various combinations of colours on a single tissue section to achieve a comparable picture of different antigens. However, the colour combinations achieved to date are not entirely satisfactory. A double immunostaining procedure is introduced here, combining the peroxidase anti-peroxidase (PAP) and immunogold staining (IGS) methods. The IGS method is a new, simple, sensitive and reliable approach to immunostaining at the light microscopic level. It was carried out in three ways. Firstly, a two-step method was used in which the second layer was goat anti-rabbit IgG adsorbed onto gold particles (GAR/Au20). Secondly, a three-step method was employed where the second layer was unlabelled goat anti-rabbit IgG and the third layer was a rabbit antibody to peroxidase adsorbed onto the gold particles (RAP/Au20) and acting as a gold-labelled IgG antigen. The third method combined the first two methods using GAR/Au20 as the second layer and RAP/Au20 as the third layer which increased the amount of bound gold and enhanced the red colour, providing a better picture. The use of gold-labelled antibodies in double immunostaining has great potential value for many studies including that of the diffuse neuroendocrine system of the gut.

Ultrastructural Localization of Four Different Neuropeptides Within Separate Populations of p-Type Nerves in the Guinea Pig Colon

The ultrastructural localization of four neuropeptides, substance P, vasoactive intestinal polypeptide, Met-enkephalin, and somatostatin, in the guinea pig colon was investigated using electron immunocytochemistry. Each peptide was localized to the large granular vesicles in separate subpopulations of p-type nerves. These nerves could often be distinguished by the size and appearance of their immunostained granular vesicle cores. Thus, vasoactive intestinal polypeptide- and somatostatin-immunoreactive vesicles contained cores that were significantly larger (p less than 0.005) than those of substance P- or Met-enkephalin-positive vesicles. In addition, vasoactive intestinal polypeptide-immunoreactive vesicle cores were less well defined and more variable in shape than those of somatostatin-positive vesicles. Substance P- and Met-enkephalin-immunoreactive vesicle cores, however, were very similar in size (p greater than 0.05) and appearance and could only be differentiated using immunocytochemistry. This study demonstrates that the four neuropeptides under investigation are present within separate, often ultrastructurally distinct, neuronal systems in the gut. The distribution of these nerves within the colon is also described.

Sequential immunostaining (gold/silver) and complete protein staining (AuroDye) on Western blots

A double staining method is described which combines immunodetection with sensitive staining of the complete electropherogram on the same membrane. The method is based on the use of Tween 20 as blocking agent, and uses immunogold/silver staining of specific antigens and gold staining of the overall protein pattern with AuroDye. This double staining makes possible the exact location of an immunodetected band within a complex protein pattern.

Microtubule‐Dependent Intracellular Motility Investigated with Nanometer Particle Video Ultramicroscopy (Nanovid Ultramicroscopy)

The movement of intracellular organelles in vertebrate tissue-cultured cells, resulting in dynamic subcellular organization and polarity by seemingly guided and purposeful transport and mutual encounters, has been the subject of relatively few thorough studies. The motility of most optically detectable organelles is saltatory. It is fundamentally different from Brownian motion and displays both similarities and essential differences with other types of intracellular organelle transport such as axonal transport, pigment granule movement, and cytoplasmic streaming in plant cells. Saltations are dependent on the presence of, and occur along, microtubules. The molecular motor is unknown and may be located in the organelle membrane, or in the microtubule wall, or in both. We developed a new approach of general applicability to probe the role of various proteins in subcellular transport. Colloidal gold particles of 20 or 40 nm diameter microinjected into living cells are invisible to the eye in the light microscope. Individual particles, can, however, easily be discerned using transmitted light a t high numerical aperture and video contrast enhancement (FIGURE 1). They appear as black dots on a white background. In epipolarization microscopy they appear as brightly staining dots on a background consisting of the interference reflection picture of the cells (FIGURE 1). Ultrastructural observation of the same cells prepared as whole mounts or in thin sections, confirmed that individual 20 nm particles could be resolved in the light microscope. The injected particles were not within membrane-lined vesicles, but free in the cytoplasmic matrix often in the vicinity of microtubules. Uncoated particles stabilized with polyethylene glycol, or particles coated with albumin were transported along microtubules in a saltatory, ATP-dependent fashion a t the same velocity and with the same frequency as endogenous organelles (FIGURE 1). Because subunit flux within microtubules is a possible mechanism, we injected gold particles coupled to a monoclonal antitubulin antibody.’ Many of these particles assumed fixed positions for several hours, often forming linear arrays. The observations show that gold particles having the same size as the smallest cellular membrane-lined organelles are transported by a microtubule-associated mechanism, as has been described for larger (250-500 nm) polystyrene and other beads microinjected in axons’ or cultured cells.’ Microtubule treadmilling does not appear to be involved. The possibility of following 2 0 4 0 nm particles, and probably even smaller ones that can be coupled to many proteins within living cells, provides a tool of wide