Permeabilization of Drosophila eggs

Vitelline membrane of Drosophila eggs were permeabilized by a brief immersion in octane. The treatment made the eggs very sensitive to desiccation and to the tonicity and the composition of the incubation medium. Permeabilized eggs were able to absorb cytological stains, metabolites, alkaloids, and antibiotics.     

On tannic acid fixation and staining

Tannic acid was found to fix and stain glycocalyx heavily. After removal of the major component of surface glycopeptides by trypsin, the surface coat was stained vaguely, and after the treatment with collagenase, the surface coat was moderately stained. It is concluded that tannic acid stained non-specific surface glycopeptides.     

In vitro activation of Drosophila eggs

Mature ovarian eggs of Drosophila can be activated by treatment with hypotonic buffers. Well-fed, 4-day-old virgin flies contain large numbers of partially dehydrated mature eggs. When these eggs are transferred to a hypotonic culture medium, the ovarian eggs swell immediately and within minutes up to 70% become impermeable. The following cellular events ensue: meiosis, which had been arrested at metaphase I, is completed and the "polar body" nuclei fuse; cortical multivesicular bodies with acid phosphatase activity appear within minutes; polar granules fragment, dissociate from mitochondria, and become associated with polysomes; finally, monosomal ribosomes move to the polysomal region of a sucrose gradient. Each of these events corresponds to the normal in vivo effects of ovulation of oocytes, whether or not they are fertilized. When ovarian eggs of a parthenogenetic strain of D. mercatorum were activated by hypotonic treatment, some eggs developed into normal embryos. The presence of high potassium, low pH, and polyethylene glycol enhanced the frequency of normal development. Thus, we suggest that the rehydration of mature oocytes, as they move from the ovary to the uterus, activates the maternal program of the oocyte.     

Microwave-accelerated fixation and lacZ activity staining of testicular cells in transgenic mice.

Microwave energy has been used in conjunction with glutaraldehyde to rapidly fix testicular samples of transgenic mice (whole tubules, individual cells, and cryosections) as a preparation for histochemical bacterial beta-galactosidase activity staining. The results demonstrate that the microwave-enhanced aldehyde fixation step is a convenient and simple adaptation for routine analyses, with almost no artifactual consequences or gross distortions in morphology at the microscopic level. The entire procedure (from sacrificing the animal to microscopic observation of the blue spermatogenic cells) can be completed in 1 h.     

Hoechst 33258 fluorescent staining of Drosophila chromosomes

Metaphase chromosomes of D. melanogaster, D. virilis and D. eopydei were sequentilly stained with quinacrine, 33258 Hoechst and Giemsa and photographed after each step. Hoechst stained chromosomes fluoresced much brighter and with different banding patterns than quinacrine stained ones. In contrast to mammalian chromosomes, Drosophia's quinacrine and Hoechst bright bands are all in centric heterochromatin and the banding patterns seem more taxonomically divergent than external morphological characteristics. Hoechst stained D. melanogaster chromosomes show unprecedented longitudinal differentiation by the heterochromatic regions; each arm of each autosome can be unambiguously identified and the Y shows eleven bright bands. The Hoechst stained Y can also be identified in polytene chromocenters. Centric alpha heterochromatin of each D. virilis autosome is composed of two blocks which can be differtiated by a combination of quinacrine and Hoechst staining. The distal block is always Q-H- while the proximal block is, for the various autosomes, either Q-H-, Q+H- or Q+H+. With these permutations of Hoechst and quinacrine staining, D. virilis autosomes can be unambiguously distinguished. The X and two autosomes have H+ heterochromatin which can easily be seen in polytene and interphase nuclei where it seems to aggregate and exclude H- heterochromatin. This affinity of fluorochrome similar heterochromatin was been seen in colcemide induced multiple somatic non-disjunctions where H+ chromosomes were distributed to one rosette and H- chromosomes were distributed to another. Knowing the base composition and base sequences of Drosophila satellites, we conclude that AT richness may be necessary but is certainly an insufficient requirement for quinacrine bright chromatin while GC richness may be a sufficient requirement for the absence of quinacrine or Hoechst brightness. Condensed euchromatin is almost as bright as Q+ heterochromatin. While chromatin condensation has little effect on Hoechst staining, it appears to be "the most important factor responsible for quinacrine brightness.' All existing data from D. virilis indicate that each fluorochrome distinct block of alpha heterochromatin may contain a single a single DNA molecule which is one heptanucleotide repeated two million times.     

The influence of fixation on staining of glycosaminoglycans in glial cells.

Cytoplasmic staining of glial cells by Alcian blue in 0.40 M magnesium chloride can be demonstrated in sections of brain tissue fixed in formol-acetic acid and in Bouin's solution. Phosphate-buffered aldehyde fixatives, whether at neutral or low pH, fail to preserve stainable material.     

Patterns of ionic current through Drosophila follicles and eggs

Large steady electrical currents traverse Drosophila follicles in vitro as well as permeabilized eggs. During the period of main follicle growth (stages 9-11), these currents enter the anterior or nurse cell end of the follicles. This inward current acts like a sodium ion influx with some calcium involvement. During the period of chorion formation (stages 12-14), foci of inward current also appear at the posterior, posterodorsal, and anterodorsal regions of follicles in vitro. In stage 14, the posterior in current acts like a chloride ion efflux. In preblastoderm eggs substantial currents continue to enter their anterior end; while weaker and less frequent ones enter their posterior end. We present models in which the currents during follicle growth are driven by the plasma membrane of the oocyte nurse cell syncitium; the external currents during choriogenesis are driven by the follicular epithelium; while the currents through the preblastoderm egg are driven by its plasma membrane. Measurements of pole-to-pole resistances and voltages across preblastoderm eggs indicate that the transcellular currents normally maintain a steady extracellular voltage gradient along the perivitelline space, with the anterior pole kept negative by perhaps 4 or 5 mV. The developmental significance of these currents is discussed.     

Tissue fixation and staining with osmium tetroxide: the role of phenolic compounds.

It has been postulated that phenol-containing areas of plant and animal tissues were osmiophilic, but proof of direct interaction between osmium tetroxide and phenolic materials, or the nature of such reactions, has been lacking. We find that, under conditions similar to those of normal tissue fixation, osmium tetroxide reacts rapidly with those phenols containing o-dihydroxy groups (including such species found in plant tissues) to give very stable chelate complexes. We conclude that these complexes are responsible for the observed electron-density in phenol-containing areas of tissue treated with osmium tetroxide, so that such phenols are indeed osmiophilic.     

A two-step method for permeabilization of Drosophila eggs

As a first step in developing a procedure for the cryopreservation of Drosophila melanogaster embryos, we have established a method for permeabilization of the eggcase and have initiated studies of the hydraulic conductivity of permeabilized embryos and the permeation of selected cryoprotective agents. The eggcase of D. melanogaster embryos has a wax layer that precludes any flux of water. A two-step procedure employing organic solvents was developed to effect removal of the wax layer with minimal deleterious effects on the embryos. Dechorionated embryos (Oregon-R strain P2, 12 to 13 hr old) were rinsed sequentially in isopropanol and hexane. After removal of solvent, embryos were held in a modified cell culture medium for further manipulation. This procedure routinely yielded 80 to 95% of the eggs permeabilized (as determined by osmotic contraction in 1 M sucrose) and 75 to 90% survival (incidence of hatching). Hydraulic conductivity of permeabilized embryos and permeation of cryoprotectants were determined using a microdiffusion chamber and computerized video microscopy. Regression analysis of the volumetric data from individual embryos yielded the Boyle-van't Hoff function FVeq = 0.124 (osm-1) + 0.541 with the standard deviations of slope and intercept (Vb) being 0.010 and 0.040, respectively. Permeabilized embryos exhibited ideal osmotic behavior over the range of 0.265 to 2.00 osm. The mean hydraulic conductivity coefficient (Lp) was 0.722 +/- 0.366 micron/(min.atm) at 20 degrees C, based on observations of contraction following a step change in concentration of Ringer's solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

The use of methyl green-pyronin staining after glutaraldehyde fixation and paraffin or araldite embedding.

Methyl green-pyronin staining has been used for localization of RNA and DNA in chick and mouse embryonic tissues and in insect larval salivary glands. Glutaraldehyde or tricholoracetic acid-lanthanum acetate (TCA-LA) was used as fixative and paraffin wax or Araldite was used as embedding medium. For good results the following are specially desirable: fixation with 2.5% glutaraldehyde, dehydration in alcohols for short time, and the use of fresh staining solutions. After TCA-LA fixation the final results are much less specific. The digestion with RNAse appears essential for the detection of RNA because pyronin does not seem to be entirely specific to RNA. The results show that glutaraldehyde a common fixative for electron microscopic work, is particularly suitable fixative for light microscopic cytochemical investigations if followed by methyl green-pyronin staining; furthermore, methyl green-pyronin staining after glutaraldehyde fixation can be carried out on Araldite sections.     

Effects of fixation, dehydration and staining on dimensions of myxosporidan

The effects of fixation, dehydration and staining on the morphological dimensions of myxo- and microsporidan spores were tested. Seven fixatives, two dehydrants and five stains were tested. Ten % formalin produced the least shrinkage and provided the best cytological detail of fixed material in both types of spores. All fixatives caused shrinkage of myxosporidan spore length and polar capsule length. Spore capsule width and polar capsule width were unaffected by 10% formalin. Ethyl alcohol caused no significant change in spore width. Microsporidan spore length shrunk with all fixatives, but spore width was generally unaffected. Dehydration, with either isopropyl alcohol or acetone, produced additional, significant shrinkage. The influence of stains on spore size was negligible. Heidenhains iron hematoxylin followed by eosin, and Mallory's analine-blue collagen stain, effectively stained myxo- and microsporidan spores.     

Differential fluorescent staining of Drosophila chromosomes with quinacrine mustard

The polytene and mitotic chromosomes of D. melanogaster, D. simulans, D. ananassae, and D. virilis were stained with the fluorescent dye, quinacrine mustard (QM). In all these species except D. ananassae, we have detected species-specific chromosomal loci which exhibit an extremely brilliant fluorescence. Most, but not all, of the brilliantly fluorescing areas are located in heterochromatic chromosome regions. Cytochemical and chemical methods have been employed to demonstrate that the brilliant fluorescence represents regions of acid labile non-covalent binding between DNA and QM whereas the moderate overall fluorescence is primarily due to covalent bonding (by alkylation) of the QM to DNA. The exact mode of binding of QM in the brilliant areas and the nature of the DNA in these areas are not known. The possible biological significance of the DNA in the brilliant regions is discussed.This study was supported by a Research Grant (GM 10499) from the National Institutes of Health, U.S. Public Health Service.This paper is dedicated with respect and affection to Professor Berwind P. Kaufmann on the occasion of his 74th birthday, April 23, 1971.