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1.
A staining method is described using thionin, for undecalcified deacrylated bone sections. RNA is stained purplish violet, allowing still active osteoblasts to be distinguished from lining cells. Staining intensity of mineralized bone is related to the degree of mineralization. Mineralizing fronts and cement lines are visualized clearly. Lamellae show an alternate pattern. Histomorphometric parameters such as osteon thickness and interstitial bone thickness can be measured without using polarized light. The mineralizing front can be assessed and expressed as a percentage of the osteoblast-covered interface between osteoid and mineralized bone. The stain is also useful for qualitative assessment of metabolic bone disease. Thionin stained sections can be kept for at least one year when stored hi the dark at 7 C. 相似文献
2.
A silver stain for the detection of nanogram amounts of tRNA following two-dimensional electrophoresis 总被引:6,自引:0,他引:6
G L Igloi 《Analytical biochemistry》1983,134(1):184-188
Three ultrasensitive protein silver-staining methods have been compared with respect to the detection of tRNA in polyacrylamide gels. The method of Sammons (D. W. Sammons, L.D. Adams, and E.E. Nishizawa (1981) Electrophoresis 2, 135-141) has been shown to have remarkable sensitivity, with a detection limit of 0.3 ng tRNA/mm2, allowing the two-dimensional fractionation of submicrogram amounts of bulk tRNA. The application of this technique to developmental and differentiation problems and other areas where the amounts of nonradioactive tRNA available are limited is anticipated. 相似文献
3.
Fang-Sheng Wu 《Planta》1987,171(3):346-357
The positively-charged fluorescent dye rhodamine 123 (r-123) specifically stains mitochondria in living plant protoplasts, suspensionculture cells, and root hairs. This dye functions as a vital stain and permits visualization of the localization, distribution and movement of the mitochondria. Dehydration of root hairs caused mitochondria to aggregate into clumps. Mitochondria were either homogenous or heterogeneous and were frequently seen to accumulate in the perinuclear regions of suspension-culture cells but not in those of protoplasts or root-hair cells. Dinitrophenol and high concentrations of ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid and KCl immediately eliminated fluorescence in r-123-stained mitochondria whereas ionomycin enhanced it. Treatment of seedlings with r-123 resulted in differential brightness of fluorescence in different tissues. Meristematic tissues, such as root and shoot tips, exhibited the brightest fluorescence. The cytotoxicity of r-123 in both germinating seedlings and suspension-culture cells was low. The specificity, sensitivity and low toxicity of r-123 should make it a useful tool in experiments designed to examine agents and conditions which affect the location, the physiological status or the viability of mitochondria.Abbreviations EGTA
ethyleneglycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid
- DAPI
46-diamidino-2-phenylindole
- r-123
rhodamine 123 相似文献
4.
Dried smears prepared from vitally stained sperm were evaluated as a method of simultaneously determining sperm viability and acrosomal morphology. A combination Fast Green FCF-Eosin B stain was used. The stained smears were examined at × 1, 250 using differential interference contrast microscopy (DIC). For comparison, the percentage of sperm with intact acrosomes was also determined from wet smears using DIC. Acrosomal morphology was not altered by the staining procedure, as the percentage of intact acrosomes was similar whether quantitated from wet or stained smears. Absence of eosinophilic staining in the acrosome was used as an indication of sperm viability. The percentage of sperm with unstained acrosomes was highly correlated with the percentage of intact acrosomes quantitated from stained smears. Thus, vital staining provided an indication of sperm viability comparable to acrosomal integrity, a highly reliable technique. The major advantages of using dried stained smears were more thorough examination of individual sperm without sperm activity interference, simultaneous evidence of sperm viability and morphology, and the opportunity to delay evaluation. In addition, diluting spermatozoa in complex or simple media with or without egg yolk or follicular fluid did not interfere with subsequent staining or acrosomal evaluation. 相似文献
5.
Inhibition of epidermal metabolism and DNA-binding of benzo[a]pyrene by ellagic acid 总被引:2,自引:0,他引:2
B J Del Tito H Mukhtar D R Bickers 《Biochemical and biophysical research communications》1983,114(1):388-394
Ellagic acid, a common plant phenol, was shown to be a potent inhibitor of epidermal microsomal aryl hydrocarbon hydroxylase (AHH) activity in vitro, and of benzo[a]pyrene (BP)-binding to both calf thymus DNA in vitro and to epidermal DNA in vivo. The in vitro addition of ellagic acid (0.25-2.0 microM) resulted in a dose-dependent inhibition of AHH activity in epidermal microsomes prepared from control or carcinogen-treated animals. The I50 of ellagic acid for epidermal AHH was 1.0 microM making it the most potent inhibitor of epidermal AHH yet identified. In vitro addition of ellagic acid to microsomal suspensions prepared from control or coal tar-treated animals resulted in 90% inhibition of BP-binding to calf thymus DNA. Application of ellagic acid to the skin (0.5-10.0 mumol/10 gm body wt) caused a dose-dependent inhibition of BP-binding to epidermal DNA. Our results suggest that phenolic compounds such as ellagic acid may prove useful in modulating the risk of cutaneous cancer from environmental chemicals. 相似文献
6.
Annie Giraud Simone Bouchilloux 《Biochemical and biophysical research communications》1983,111(2):353-359
The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [3H] glucosamine and [35S] , enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [3H] label and, when cells organized into follicles, of the proportion of cell-associated [3H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates. 相似文献
7.
When the conditions for detecting proteins by ammoniacal silver staining (B. R. Oakley, D. R. Kirsch, and N. R. Morris (1980) Anal. Biochem. 105, 361-363.) following gel electrophoresis were varied, it was noted that glutaraldehyde pretreatment was necessary for maximal staining, which could not be explained simply as the result of "fixation." Further studies indicated that glutaraldehyde enhancement of protein staining with this silver reagent was probably due to oxidation of the aldehyde groups by silver ions, resulting in metallic silver depositions within the gel which act as nucleation sites for additional metallic silver localization in the protein bands upon the addition of formaldehyde developer. This proposed mechanism is consistent with the Tollen's reaction, as well as some aspects of the photographic process. Consistent with this notion, silver-staining intensities are directly related to mole percentage lysine of various standard proteins. 相似文献
8.
Characteristics of the volume- and chloride-dependent K transport in human erythrocytes homozygous for hemoglobin C 总被引:2,自引:0,他引:2
Carlo Brugnara 《The Journal of membrane biology》1989,111(1):69-81
Summary In human red cells homozygous for hemoglobin C (CC), cell swelling and acid pH increase K efflux and net K loss in the presence of ouabain (0.1mm) and bumetanide. We report herein, that K influx is also dependent on cell volume in CC cells: cell swelling induces a marked increase in the maximal rate (from 6 to 18 mmol/liter cell × hr) and in the affinity for external K (from 77±16mm to 28±3mm) of K influx. When the external K concentration is varied from 0 to 140mm, K efflux from CC and normal control cells is unaffected. Thus, K/K exchange is not a major component of this K movement. K transport through the pathway of CC cells is dependent on the presence of chloride or bromide; substitution with nitrate, acetate or thiocyanate inhibits the volume- and pH-dependent K efflux. When CC cells are separated according to density, a sizable volume-dependent component of K efflux can be identified in all the fractions and is the most active in the least dense fraction. N-ethylmaleimide (NEM) markedly stimulates K efflux from CC cells in chloride but not in nitrate media, and this effect is present in all the fractions of CC cells separated according to density. The persistence of this transport system in denser CC cells suggests that not only cell age, but also the presence of the positively charged C hemoglobin is an important determinant of the activity of this system. These data also indicate that the K transport pathway of CC cells is not an electrodiffusional process and is coupled to chloride. 相似文献
9.
Intensity of bacterial flagella staining using a modified silver stain was increased by aging the mordant for one week at room temperature. The use of aged mordant increased the apparent diameters of stained flagella and resulted in a darker stain. The mordant remained stable for at least four months at room temperature. The staining protocol presented allows application to liquid or solid cultures. 相似文献
10.
Evaluation of Rhodamine 123 As A Probe For Monitoring Mitochondrial Function In Trypanosoma Brucei Spp. 总被引:3,自引:0,他引:3
ALAN A. DIVO CURTIS L. PATTON ALAN C. SARTORELLI 《The Journal of eukaryotic microbiology》1993,40(3):329-335
ABSTRACT. Rhodamine 123, a membrane potential-specific dye, has been evaluated as a probe to monitor the function of the mitochondrion in long slender bloodstream and procyclic trypomastigotes of several Trypanosoma brucei spp. By epifluorescence microscopy, mitochondrial development has been followed in long slender bloodstream and procyclic organisms stained with rhodamine 123. to photograph stained long slender bloodstream forms, it was necessary to develop a method to completely immobilize viable organisms. In both parasite forms, as the cell cycle progressed, the mitochondrion developed from a thread-like structure to a highly branched organelle. A dramatic reorganization occurred preceding cytokinesis to produce two progeny thread-like structures which were partitioned into newly formed daughter cells. the organelle within the long slender trypomastigote was found to stain optimally at 0.3 μ/ml of rhodamine 123, while the procyclic form required 3.0 μ/ml. the results suggest that the plasma membrane potential is higher in the long slender parasite than in the procyclic form. the effects of inhibitors that disrupt mitochondrial function were examined in long slender and procyclic parasites, and some of these agents were shown to affect rhodamine 123 accumulation and retention. In long slender trypomastigotes the trypanosome alternative oxidase does not appear to be coupled to proton pumping, whereas in procyclic organisms the effects of inhibitors indicate that this oxidase may be coupled to a pathway that is branched preceding an antimycin A1 -sensitive site. 相似文献