1. A rapid, mild method for isolating nuclear membranes from isolated rat liver nuclei is described. The method employs a double digestion of nuclei with a low level of DNAase I at approximately 0.1 mM Mg2+ and slightly alkaline pH, to release the membranes.2. Electron microscopy shows excellent preservation of the nuclear membrane morphology. Large membrane fragments consisting of a double membrane with ribosomes adhering to the outer layer are produced. The layers are connected at nuclear pores, which have distinct annular subunits and occasional central granules. Contamination by other subcellular structures is minimal.3. The recoveries of nuclear protein, DNA, RNA and phospholipid in the membrane are approximately 8O/,, I -3O/,, So/, and 550/,, respectively. The DNA associated with the membrane does not appear to result from the reassociation of released DNA. The enzymic content of the membrane is similar to that of the microsomes. It is concluded that the nuclear membrane is the sole nuclear location of glucose-6-phosphatase.4. About lo/, of the total nuclear DNA polymerase was recovered associated with the nuclear membrane. By three criteria this enzyme was identical with pure authentic nuclear DNA polymerase. This finding is discussed against the background of the suggestions that the nuclear membrane is involved in DNA replication.A role for the nuclear membrane has been postulated in the widely differing fields of DNA replication [I, 21, the organisation of chromatin [ 3 ] , the formation of the herpes virus nucleocapsid envelope[4], nucleo-cytoplasmic exchange of macromolecules and nuclear respiration [ 5 ] .InveAtigation of some of these postulated functions would be considerably facilitated by suitable membrane preparations. Existing preparative methods are slow, taking a t least 12 h from the isolation of the nuclei and involve either sonication or high salt extraction or both r6-91. Sonication causes membrane fragmentation and in some cases fusion between different membranes [lo], while high salt is likely to extract many proteins associated with the membrane.We describe here a rapid, mild method of isolating nuclear membranes from the isolated nuclei which takes approximately 80 mjn and avoids both sonication and high salt extraction. The isolated membranes have been characterised chemically, biochemically and by microscopy and have the features expected for the nuclear membrane.There exists considerable controversy as to the site of nuclear DNA replication [1,2,11,12]. It, has been suggested that the association of a DNA polymerase activity with the nuclear membrane indicated this to be the site of DNA replica.tion [13]. I n a previous publication we found that newly replicated DNA was not concentrated in the nuclear membrane fraction of regenerating rat liver nuclei and so concluded that this was probably not the site of all DNA replication [Ill. We report here that the small proportion of the total nuclear DNA polymerase found associated with the nuclear membrane is identical by three criteria ...

We report here the results observed when tubulin fluorescence immunohistochemistry is performed upon dissociated cultures of nervous tissue, principally of chick embryo spinal cord. When fixation includes nonpolar solvents or detergents, a uniform fluorescence is seen in neuron perikarya (with the exception of their nuclei), and the processes to which they give rise. Fixation with formaldehyde or glutaraldehyde alone, however, results in a discontinuous staining of neurites, and a less regular staining of their perikarya. The former pattern of response can be elicited if aldehyde fixation is followed by exposure to non-polar solvents. Such results are obtained both in thinly spread regions of the cultures, where neurons and their processes can easily be seen, and in the cell aggregates that also characterise them. Possible interpretations of these results are discussed.