Detection of impervious tissue in tree bark with selective histochemistry and fluorescence microscopy

Use of conventional histochemical tests in conjunction with fluorescence microscopy has validated the concept of impervious tissue in the bark of trees. Application of phloroglucinol + HCl or toluidine blue O selectively quenched lignin autofluorescence and allowed visualization of intracellular suberin lamellae previously undetected. Fluorescence of intracellular lamellae was quenched with Sudan black B and enhanced with Sudan IV thus providing evidence for the suberized nature of a tissue heretofore regarded as nonsuberized.     

Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol

Polyethylene glycol (400) with 90% glycerol (aqueous) is introduced as an efficient solvent system for lipid stains. Various lipid-soluble dyes were dissolved in this solvent system and tested for their intensity, contrast, and specificity of staining of suberin lamellae in plant tissue. The stability (i.e., lack of precipitation) of the various staining solutions in the presence of fresh tissue was also tested. When dissolved in polyethylene glycol-glycerol, Sudan red 7B (fat red) was the best nonfluorescent stain and fluorol yellow 088 (solvent green 4) was an excellent fluorochrome. These two dyes formed stable staining solutions which efficiently stained lipids in fresh sections without forming precipitates. Estimations of the solubilities of these dyes in the solvent compared with their solubilities in lipids of various chemical types indicated that they should both be effective stains for lipids in general.     

植物细胞壁组织化学定位染色方法和技术的比较研究

利用光学和荧光显微镜比较研究几种植物细胞壁组织化学定位染色方法和技术,结果表明：（1）硫酸消化法和硫酸氢黄连素-苯胺兰对染法研究凯氏带,对取材时间和部位要求高,建议两种方法配合使用,可相互印证是否具凯氏带;（2）苏丹7B染色法,蓝色激发光下不染色和硫酸氢黄连素-苯胺兰对染研究细胞壁栓质层3种方法中,不染色蓝色激发光下结果比苏丹7B染色法敏感显色,但苏丹7B染色法在普通光学显微镜下观察较为便捷;（3）木质化细胞壁染色方法中硫酸氢黄连素-苯胺兰对染法比间苯三酚-盐酸染色法易显色观察;（4）甲苯胺兰快速染色细胞壁取代常规苏丹Ⅲ/Ⅳ法,细胞边界和层次更清楚。     

Evaluation of intracellular lipids by standardized staining with a Sudan black B fraction.

The selective staining of neutral lipids in Human Amnion cells in tissue culture was achieved using a particular fraction of the lipid dye, Sudan black B and a standardized staining procedure. The fraction, termed SBB-I, was isolated by thin-layer chromatography. The cytophotometric assessment of intracellular neutral lipids, stained with SBB-I, is described and applied to the study of changes in granulocytic neutral lipids in leukemia.     

Structural organization and possible functional role of annulate lamellae containing cytoplasmic pores

This review is devoted to annulate lamellae, a specific compartment of endoplasmic reticulum that occurs, presumably, in actively growing and rapidly dividing cells (oocytes, embryonic and tumor cells). We summarized both earlier and recent data on the dustribution of annulate lamellae in various cell types, on their morphology, and the distribution of interaction with intracellular structures at various treatments. As the annulate lamellae contain cytoplasmic pore complexes, a special attention was paid to their relation with nuclear pores. Possible functions of the annulate lamellae in intracellular processes and, particularly, in nuclear envelope assembly, are discussed.     

Chromatography and Biological Stains: III. Comparison of the Fat Staining Efficiency of Fractions of Commercial Sudan III Separated by Column Chromatography

Fractions of commercial Sudan III which were separated by column chromatography were compared for fat staining efficiency. Paraffin sections of chromated mouse liver tissue and sections of both fresh and formalin-fixed rat liver tissue or Musca domestica larvae, cut with the freezing microtome, were used. Evidence is presented that a sample of very highly purified Sudan III has no ability to render a fat stain in fresh, formalin-fixed or chromated tissue. However, certain other fractions from the commercial sample, some completely devoid of Sudan III, had good staining characteristics. It is concluded that some substance or substances, other than Sudan III, is responsible for the staining action of the commercial dye.     

Movement of lipolytic products to mitochondria in brown adipose tissue of young rats: an electron microscope study

Lipolysis occurred and lamellar structures with a periodicity of 40 A developed in glutaraldehyde-fixed brown adipose tissue of suckling rats when the tissue was incubated at 25 degrees C. The lamellar structures were found in capillaries, associated with chylomicrons, in intracellular channels of capillary endothelium, in extracellular space, and in channels near lipid droplets in adipocytes in tissue of fed rats injected intravenously with chylomicrons. They were also found in channels near mitochondria and inside mitochondria in adipocytes in incubated-fixed tissue of rats exposed to 4 degrees C for 2 hr or unsuckled overnight. In addition, aqueous spaces developed adjacent to lipid droplets in incubated tissue of cold-exposed and unsuckled rats. Development of lamellar structures under conditions causing lipolysis and accumulation of fatty acids in fixed tissue indicated the lamellae were composed primarily of fatty acids. We conclude that fatty acids formed by lipolysis of chylomicrons in tissue from fed rats accumulated in a continuum of the outer leaflets of cell membranes extending from capillary lumen to lipid droplets of adipocytes, and fatty acids formed by lipolysis of intracellular lipid in tissue from cold-exposed or unsuckled rats accumulated mostly in a continuum extending from lipid droplets to the interior of mitochondria. When fatty acids overcrowded the continuum in fixed tissue, they formed lamellar extensions of the continuum at different sites along its course through the tissue.     

Vinblastine-induced formation of annulated lamellae in SV3T3 cells

SV40-transformed 3T3 cells formed microtubular crystals in the cytoplasm when treated with vinblastine sulfate. Annulated lamellae, an unusual intracellular organelle found in some actively proliferating cell types, appeared concurrently with the formation of microtubular crystals. Although crystals were also seen in non-transformed 3T3 cells treated with vinblastine, annulated lamellae were not found.     

Effect of phosphomolybdic acid on the binding of Sudan black B

Paraffin sections of tissues fixed in absolute alcohol or Carnoy's fluid were mordanted in a 1% aqueous solution of phosphomolybdic acid, stained in saturated solutions of Sudan black B, acetylated Sudan black, various solvent and basic dyes in 70% ethyl alcohol for 5 min at room temperature, dehydrated in alcohol and covered in Permount. Sudan black B and other dyes with basic groups stained basement membranes, reticulum and collagen fibers intensely. Acetylated Sudan black, Sudan IV and oil red 0 did not color any tissue structures. Control sections, without pretreatment, did not bind Sudan black B. These findings indicate interaction between basic groups of the dye and free acid groups of phosphomolybdic acid.     

Propylene and Ethylene Glycol as Solvents for Sudan IV and Sudan Black B

Propylene or ethylene glycol is recommended as a solvent for Sudan IV and Sudan black B to replace the commonly used alcohol-acetone mixtures for general lipid staining in tissue sections. Either glycol is used as a dehydrating agent, dye solvent, and differentiating solution. They offer the advantages of a stable solution, inert with respect to solubilities of lipid material in it, and excellent control of differentiation without loss of dye from lipid particles. Sections remain pliable and are not shrunken by the glycols. Counterstains may be used after staining with Sudan IV but are generally not necessary after staining with Sudan black B. With the use of propylene glycol as a solvent, Sudan IV appears to equal the staining ability of Sudan black B as regards the type of lipid material detected, and the choice of dye to be used would depend on the color contrast desired.     

Gill ultrastructure of the Pacific hagfish Eptatretus stouti

At the gross anatomical level, hagfish gills show unusual features not seen in any other fish gills. Our study was undertaken to determine if peculiarities also characterize the microscopic anatomy and ultrastructure of hagfish gills. To the contrary, branchial respiratory lamellae of Pacific hagfish were found to resemble the lamellae of lampreys, elasmobranchs, and teleosts, often down to the finest subcellular details. As in other fish, hagfish lamellae are lined by epithelium containing pavement cells with organelles indicative of a secretory function, basal cells showing undifferentiated cell features, and branchial ionocytes. The ionocytes are identical to chloride cells of teleosts in cytostructure, distribution, and abundance. There are pillar and marginal capillaries in hagfish gill lamellae. Pillar cells contain bundles of 5-nm microfilaments, and they associate with collagen columns as in other fish. Hagfish pillar cells do exhibit odd features, however: They cluster (groups of up to nine were seen), and their extracellular collagen columns are rarer than in other fish gills (averaging only two columns per three pillar cells). Other special features of hagfish gills are the following: lipid droplets and smooth endoplasmic reticulum are well developed in all cell types; pavement cells secrete a lipomucous product (stains with periodic acid-Schiff, Alcian blue, and Sudan black B); and goblet cells are absent. The presence of "chloride cells" in hagfish is puzzling, as hagfish body fluids are iso-osmotic to seawater and there is no need to osmoregulate for sodium chloride; the ionocytes contain carbonic anhydrase, suggesting a function in acid/base regulation.     

A study of several blue-green algae in the electron microscope

Summary All of the three blue-green algae, Anabaena cylindrica, Mastigocladus laminosus and Nostoc muscorum are characterized by the presence of multi-layered envelopes (sheath, wall and plasma membrane), photosynthetic lamellae and a variety of intracellular granules. Sections of heterocysts of Anabaena cylindrica showed the presence of an internal membrane system as well as lamellae. An unusual feature of the structure of Nostoc muscorum was the presence of densely stained intracellular membranes or lamellae. The results emphasize the variability in appearance of the internal structure of the blue-green algae and point to the need for detailed investigations of the influence of change in physiological environment on the anatomy of these organisms.     

Hsp70 and a 54 kDa protein (Osp54) are induced in salmon (Salmo salar) in response to hyperosmotic stress.

Hsp70 and a 54 kDa osmotic stress protein (osp54) were induced in isolated tissues of anadromous Atlantic salmon (Salmo salar) upon exposure to hyperosmotic conditions. Incubation of branchial lamellae, hepatic tissue, and erythrocytes in medium supplemented with 200-600 mM NaCl dramatically reduced protein synthesis. Although general protein synthesis remained depressed following transfer of tissues from 450 mM supplemental NaCl to iso-osmotic medium, hsp70 was prominently induced in branchial lamellae and hepatic tissue. Accumulation of hsp70 mRNA and a decrease in actin mRNA suggest preferential upregulation of the hsp70 gene. Induction of osp54 was observed in branchial lamellae and erythrocytes, but not in hepatic tissue, during exposure to 75-125 mM supplemental NaCl. Use of glycerol in place of NaCl to create hyperosmotic conditions stimulated induction of hsp70 in branchial lamellae. Substitution with mannitol resulted in induction of osp54 in both branchial lamellae and erythrocytes. The solute-specific and temporal patterns of response suggest that hsp70 and osp54 might function in concert to restore osmotic homeostasis and renature proteins destabilized or denatured during the early stages of osmotic shock.     

Some Staining Technics for Guayule Studies

For staining tissues and secretions in guayule studies the following dyes and dye combinations have been found satisfactory: rubber in plant sections, Sudan IV; plant sections (rubber and tissues) and ground tissues, Poirrier's blue, Sudan IV and titan yellow; rubber extraction films (with tissue contamination), oil blue NA, safranin O and titan yellow; resin extraction films, Bismark brown and oil blue NA; chopped plants (milling studies), safranin O and Sudan IV. For microscopic studies, sections and ground tissues are pretreated with KOH and a bleaching agent prior to staining.     

STAINING AND ACID PHOSPHATASE REACTIONS OF SPHEROSOMES IN PLANT TISSUE CULTURE CELLS

Spherosomes in plant tissue culture cells from normal sunflower stems and sunflower crown gall tumors reacted similarly to several nonfluorescent and fluorescent lipid dyes. Sudan IV and black B were good selective spherosome stains. The lipid fluorochromes, Nile blue and 3, 4-benzpyrene were excellent selective spherosome stains and visualized the smallest particles more readily than did Sudan IV. Spherosomes could not be seen in tissues stained with Sudan IV or 3,4-benzpyrene after ether-alcohol extraction. Acid phosphatase was detected on the spherosomes in both normal and tumor tissues using the lead sulfide precipitation and the post-incubation coupling procedures.     

Inhibition of Sudan I genotoxicity in human liver-derived HepG2 cells by the antioxidant hydroxytyrosol

The chemoprotective effect of hydroxytyrosol (HT) against Sudan I-induced genotoxicity was investigated in a human hepatoma cell line, HepG2. The comet assay and micronucleus (MN) assay were used to monitor genotoxicity. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe, 2,7-dichlorofluorescein diacetate (DCFH-DA). The levels of oxidative DNA damage and lipid peroxidation were estimated by immunocytochemistry analysis of 8-hydroxydeoxyguanosine (8-OHdG) and by measuring levels of thiobarbituric acid-reactive substances (TBARS), respectively. Intracellular glutathione (GSH) level was estimated by fluorometric methods. The results showed that HT significantly reduced the genotoxicity caused by Sudan I. Furthermore, HT ameliorated lipid pexidation as demonstrated by a reduction in TBARS formation and attenuated GSH depletion in a concentration-dependent manner. It was also found that HT reduced intracellular ROS formation and 8-OHdG level caused by Sudan I. These results strongly suggest that HT has significant protective ability against Sudan I-induced genotoxicity.     

Acute morphological changes of palisade cells ofSaintpaulia leaves induced by a rapid temperature drop

Early changes in fine structures of intracellular organelles ofSaintpaulia leaves exhibiting chilling-induced degeneration of photosynthetic activities were investigated by electron microscopy. As early as 3 min after exposure to 10C water, thylakoid lamellae in the palisade cells showed extensive disarrangement, especially in intergrana lamellae. Simultaneously, other organelles showed drastic morphological changes, including the condensation of chromatin in the nucleus.     

Lamellar Thickness and Stretching Temperature Dependency of Cavitation in Semicrystalline Polymers

Polybutene-1 (PB-1), a typical semicrystalline polymer, in its stable form I shows a peculiar temperature dependent strain-whitening behavior when being stretched at temperatures in between room temperature and melting temperature of the crystallites where the extent of strain-whitening weakens with the increasing of stretching temperature reaching a minima value followed by an increase at higher stretching temperatures. Correspondingly, a stronger strain-hardening phenomenon was observed at higher temperatures. The strain-whitening phenomenon in semicrystalline polymers has its origin of cavitation process during stretching. In this work, the effect of crystalline lamellar thickness and stretching temperature on the cavitation process in PB-1 has been investigated by means of combined synchrotron ultrasmall-angle and wide-angle X-ray scattering techniques. Three modes of cavitation during the stretching process can be identified, namely “no cavitation” for the quenched sample with the thinnest lamellae where only shear yielding occurred, “cavitation with reorientation” for the samples stretched at lower temperatures and samples with thicker lamellae, and “cavitation without reorientation” for samples with thinner lamellae stretched at higher temperatures. The mode “cavitation with reorientation” occurs before yield point where the plate-like cavities start to be generated within the lamellar stacks with normal perpendicular to the stretching direction due to the blocky substructure of the crystalline lamellae and reorient gradually to the stretching direction after strain-hardening. The mode of “cavitation without reorientation” appears after yield point where ellipsoidal shaped cavities are generated in those lamellae stacks with normal parallel to the stretching direction followed by an improvement of their orientation at larger strains. X-ray diffraction results reveal a much improved crystalline orientation for samples with thinner lamellae stretched at higher temperatures. The observed behavior of microscopic structural evolution in PB-1 stretched at different temperatures explains above mentioned changes in macroscopic strain-whitening phenomenon with increasing in stretching temperature and stress-strain curves.     

Quench cooling and ice crystal formation in biological tissues

The rates of cooling of tissue packages quenched in liquid nitrogen were investigated using microthermocouples. By assembling tissue packages from a standard 200-μm tissue slice a microthermocouple could be positioned at different depths within the package. Results showed that for a given mass of tissue the rates of cooling at different depths were the same. When the tissue mass was varied the rates of cooling at a fixed depth decreased with increasing tissue mass.The ice crystal formations produced when tissues are quenched in liquid nitrogen were investigated using freeze substitution. Assemblies of rabbit cornea of different thicknesses were quenched in liquid nitrogen and freeze substituted. The size of the ice crystal cavities produced during the quenching increased with increasing tissue mass, exhibiting a saturation size for the larger tissue masses. There was no obvious size distribution of the ice crystal cavities across the thickness of the corneas.The results suggest an “isotherm” model for the quenching conditions used in these experiments, there being small or negligible temperature gradients through the tissue which uniformly cools at a fixed rate.     

ELECTRON MICROSCOPY OF THE PACINIAN CORPUSCLE

The Pacinian corpuscle has a framework of cytoplasmic lamellae arranged concentrically in the outer zone, and bilaterally in the core. Between these is an intermediate growth zone. The inner core shows an unexpected complexity in that its component lamellae are arranged in two symmetrical groups of nested cytoplasmic sheets. Longitudinal tissue spaces form clefts separating the two groups. The perikarya of the core lamellae lie in or near the intermediate growth zone, and send arms into the clefts. The arms then branch and terminate as lamellae which interdigitate with those of neighboring cells. The single nerve fiber loses its myelin sheath just before it reaches the inner core but retains its Schwann cell cytoplasmic covering for a short additional distance. The Schwann sheath is not continuous with the lamellae of the inner core. Inside the core the fiber contains a striking circumferential palisade of radially disposed mitochondria. The fiber does not arborize. Vascular capillaries penetrate the hilar region of the corpuscle only as far as the myelinated sheath of the nerve, and they have not been seen elsewhere in the corpuscle. There is direct continuity between the clefts of the core and tissue spaces in the vicinity of the capillaries. It is likely that this provides a route whereby metabolites reach the active nerve ending, as well as the cells of the growth zone. The outer zone consists of at least 30 flattened concentric cytoplasmic lamellae separated from one another by relatively wide fluid-filled spaces. Collagenous fibrils are present, particularly on the outer surface of lamellae, and tend to be oriented circularly. The girdle of proliferating cells constituting the growth zone, which is prominent in corpuscles from young animals, is the layer from which the outer lamellae are derived. Osmotic forces probably elevate the lamellae, and maintain turgor pressure.