A new one-step method for the cytochemical localization of ouabain-sensitive,potassium-dependent p-nitrophenylphosphatase activity

Summary A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0°–4° C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.This study was supported partly by grants-in-aid for scientific reasearch from the Ministry of Education, the Japanese Government (Nos. 244016 and 337001)Part of this paper was presented at the 19th Annual Meeting of the Japan Society of Histochemistry and Cytochemistry held in Gifu, November 1–2, 1978 (Mayahara et al. 1979a), and the 35th Annual Meeting of the Japanese Society of Electron Microscopy, held at Takarazuka, May 23–25, 1979 (Mayahara et al. 1979b)     

A simple two-step labeling procedure for ultrastructural localization of cell surface anionic sites

We propose a new method for ultrastructural localization of cell surface anionic sites. The method consists of sequential interaction of aldehyde-fixed cells with a polycationic reagent, poly-L-lysine (PL), followed by secondary interaction with a negatively charged marker, ferritin. By use of PL of low molecular weight (4000) on aldehyde-pre-fixed red blood cells and macrophages, the reaction resulted in binding of ferritin particles to cell surface anionic sites with a density distribution resembling that of cationized ferritin (CF). The density of the attached ferritin molecules increased in direct correlation with the MW of PL used. The primary PL interaction can be carried out at low pH (less than 2), thus restricting the labeling mainly to membrane-bound sialyl residues.     

Iodide,thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity

Summary The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation in vitro and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm.It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation in vitro is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections.The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN^– or CNS^–. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN^–, should be substituted for I^– in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.This work was supported by the grant of Research association of Slovenia and by the NIH grant PL 480 N° 02-008-N     

A simple, one-step chromatographic procedure for the purification of lysozyme

A rapid method for the purification of lysozyme (mucopeptide N-acetyl-muramoylhydrolase, EC 3.2.1.17) from hen egg-white has been devised. It was that gel filtration chromatography on agarose columns can be used selectively to purify lysozyme, due to the fact that this protein interacts with the agarose matrix and elutes later than the corresponding total volume for the column. Thus, lysozyme is directly obtained in a relatively pure form and with a high specific activity. In principle, this simple method can be used to prepare lysozymes from other sources.     

Iodide, thiocyanate and cyanide ions as capturing reagents in one-step copper-thiocholine method for cytochemical localization of cholinesterase activity.

The necessity of the presence of iodide in Cu-ThCh reaction was investigated by following the precipitate formation "in vitro" and by evaluating the ultrastructural localization of the precipitate in sympathetic ganglion cells of the frog and in the end-plate regions of the rat diaphragm. It was found that thiocyanate or cyanide is the only anion that can be substituted for iodide as the capturing agent in precipitation. The optimal concentration in the preincubation and incubation media of any one of the three anions is from 2 to 5 mM. At a concentration below 1 mM precipitation "in vitro" is considerably delayed as a result of which in electron microscopy diffusion artefacts appear in tissue sections. The unconverted primary precipitate obtained in the presence of iodide had been used for ultrastructural localization of ChE activity and now this use has been extended to precipitates obtained in the presence of CN- or CNS-. Better-quality localization in the presence of either one of the latter anions suggests that they, and particularly CN-, should be substituted for I- in the one-step Cu-ThCh method for the cytochemistry of cholinesterases.     

Ultrastructural and cytochemical comparison between calf and cow oocytes

The use of prepubertal females (calves) to obtain oocytes for in vitro fertilization (IVF) programs, is being analyzed currently. This will increase the availability of female oocytes and will allow a reduction of the interval between generations. Differentials in the development capability of calf and cow oocytes have been assessed by different authors, establishing several ultrastructural and metabolic differences between them. This paper analyzes the morphometric and cytochemical differences between calf and cow oocytes through microscopic techniques. The oocytes morphologically classified as good are processed for electron microscopy a) in Epon 812 epoxy resin for morphometric analysis or b) in low temperature Lowycril K4M resin for cytochemical evaluation using Con A, GS, LPA, UEA, and WGA lectins marked with colloidal gold as probes. Calf oocytes show a greater density of microvilli on their surface and a greater number of endocytosic vesicles than those of the cow. On the other hand, cow oocytes show a larger superior mitochondrial population. In the cumulus cells it can be seen that calf oocytes have a greater volume of lipid droplets. Cytochemical analysis shows that calf oocytes have lectin marking restricted to the plasmic membrane, highlighting the presence of LPA. In cow oocytes, lectin marking can be seen both on the plasmic membrane and in the vacuoles, in both cases, with the LPA highlighted. In the zona pellucida of calf and cow oocytes, the same sugars appear (GS, LPA, WGA), and marking with LPA is more extensive in cow oocytes.     

A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase

A two-step procedure for the purification of 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGDH) from sheep liver is described. The enzyme is directly bound to cellulose phosphate by batch extraction and eluted with a linear salt gradient. Purification is completed by affinity chromatography using NADP(+)-agarose. The result is 6-PGDH of high purity, greatly increased yield, and the highest specific activity yet achieved, with a significant reduction in the purification time.     

A two-step centrifugation procedure for the purification of sheep erythrocyte antigen-binding cells.

A two-step centrifugation procedure has been developed to isolate greater quantities of highly purified sheep erythrocyte antigen-binding cells (ABC) than previously possible. The first step involves partially separating sheep erythrocyte rosettes from unrosetted lymphocytes by their difference in buoyant density on Ficoll-Hypaque. Subsequent passage through a linear 5 to 10% Ficoll gradient produces further purification of rosettes by sedimentation velocity. Approximately 4.5 X 10(6) ABC can be obtained at 50 to 100% purity from 10(9) immune spleen cells (5 days post-immunization) and 1 X 10(5) ABC at 20 to 40% purity from 10(9) nonimmune spleen cells. The purified ABC from 5-day immune animals are 80 to 90% B cells and 10 to 20% T cells, and represent between 30 and 40% of the original ABC in the spleen cell population. Less than 0.2% of the purified ABC are plaque-forming cells (PFC) and less than 2% have intracellular immunoglobulin (Ig) or J chain. The quantities of ABC obtained are sufficient for investigating biochemical parameters of antigen-induced lymphocyte activation and for direct analysis of the surface isotypes found on antigen-binding cells after immunization.     

A one-step, low background coomassie staining procedure for polyacrylamide gels

Present Coomassie staining procedures require hours of destaining and/or have high backgrounds. This one-step staining procedure is easier, gives lower background with no loss in sensitivity, uses less chemicals, requires less time, and can be followed by silver stain if increased sensitivity is desired after analyzing the results.     

A one-step procedure for isolation and resolution of the Phaseolus vulgaris isolectins by affinity chromatography

A new fast-working one-step method for the resolution of the Phaseolus vulgaris isolectins is described which requires inexpensive materials.     

A two-step procedure for automatic and accurate segmentation of volumetric CLSM biofilm images

This paper presents a robust two-step segmentation procedure for the study of biofilm structure. Without user intervention, the procedure segments volumetric biofilm images generated by a confocal laser scanning microscopy (CLSM). This automated procedure implements an anisotropic diffusion filter as a preprocessing step and a 3D extension of the Otsu method for thresholding. Applying the anisotropic diffusion filter to even low-contrast CLSM images significantly improves the segmentation obtained with the 3D Otsu method. A comparison of the results for several CLSM data sets demonstrated that the accuracy of this procedure, unlike that of the objective threshold selection algorithm (OTS), is not affected by biofilm coverage levels and thus fills an important gap in developing a robust and objective segmenting procedure. The effectiveness of the present segmentation procedure is shown for CLSM images containing different bacterial strains. The image saturation handling capability of this procedure relaxes the constraints on user-selected gain and intensity settings of a CLSM. Therefore, this two-step procedure provides an automatic and accurate segmentation of biofilms that is independent of biofilm coverage levels and, in turn, lays a solid foundation for achieving objective analysis of biofilm structural parameters.