Vapor fixation for immunocytochemistry and X-ray microanalysis on cryoultramicrotome sections.

Several organic and inorganic vapor fixatives have been tested for their ability to stabilize the ultrastructure of freeze-dried thin cryosections. The vapors from osmium tetroxide and dry formaldehyde gave a good preservation of the ultrastructure. Fixation in formaldehyde vapor preserved the immunoreactivity of alpha-amylase in exocrine pancreas, as was demonstrated with an indirect labeling technique using anti-alpha-amylase and protein A-gold. A major advantage of the use of vapor fixation is that cryosections from a specimen of fresh-frozen tissue can be used for immunocytochemistry as well as for X-ray microanalysis, as was demonstrated for the exocrine pancreas. This opens the possibility of localizing atomic species (X-ray microanalysis) and molecular species (immunocytochemistry) at the subcellular level on thin cryosections from the same tissue block.

Transfer, observation and analysis of frozen hydrated specimens.

Hitherto, the observation of frozen hydrated specimens in transmission electron microscopes has been inhibited due to the technical difficulties experienced in transferring the specimen to the microscope and maintaining it at a low temperature during observation. This has resulted in loss of the primary advantage of freezing since the frozen water had to be removed from the specimen before it could be introduced into the electron microscope. The cryo-transfer system overcomes these objections and provides a means to transfer frozen hydrated specimens from any preparation equipment into the microscope without ice condensation on the specimen. The cryo-transfer system consists of a cryo-transfer unit, a cryo-specimen holder and a temperature control unit.

High-performance liquid chromatographic separation and photometric detection of phospholipids.

A rapid and efficient method for the separation of (phospho)lipids by high-performance liquid chromatography using n-hexane-2-propanol-water mixtures as the solvent system is described. The lipid separation occurs on silica gel columns and the individual components are monitored directly by UV absorption at 206 nm. Of a total lipid extract from erythrocytes as well as suboesophageal ganglia of the snail Helix pomatia, a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are partly separated under these circumstances. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some instances, as is shown for phosphatidylcholines and sphingomyelins.

High performance liquid chromatographic separation and direct ultraviolet detection of phospholipids.

A fast and efficient method for the separation of (phospho)lipids by high performance liquid chromatography using n-hexane, 2-propanol, water mixtures as the solvent system is described. The lipid separation occurs on a LiChrosorb Si-60 (10 micron) column and the individual components are monitored directly by ultraviolet absorption at 206 nm. Of a total lipid extract from erythrocytes a complete separation is achieved of cholesterol, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, lysophosphatidylcholine and lysophosphatidylethanolamine, whereas phosphatidylcholine and sphingomyelin are only partly separated under these circumstances. Furthermore, a mixture of synthetic phospholipids, i.e. 1,2-dilauroyl-sn-glycero-3-phosphatidic acid, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-1'-sn-glycerol and 1,2-dioleoyl-sn-glycero-3-phosphocholine has been completely resolved. In addition to separation of phospholipids in different classes, separation of molecular species can also be achieved in some cases, as is shown for 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine and 1,2-didocos-13'-cis-enoyl-sn-glycero-3-phosphocholine.

Absence of cell communication for fluorescein and dansylated amino acids in an electrotonic coupled cell system.

Quantitative evaluation of the diffusion process of sodium fluorescein and dansylated amino acids in the salivary gland of the larvae of Drosophila hydei reveals that the differences in specific permeability between the junctional and nonjunctional membranes, as found for small ions, do not apply to the fluorescent probes. There are no significant differences between the permeability properties for the different dansylated amino acids tested, and the same properties are found for sodium fluorescein.