A clearing technique for histochemical location of GUS activity in pollen tubes and ovules ofLycopersicon

We describe a protocol to locate GUS activity, remove browning due to polyphenolics and clear floral tissue fromLycopersicon species. Suggestions are given to adapt this protocol for samples from other species.     

The use of Herr four-and-a-half clearing fluid for the rapid microscopic examination of thick sections of normal and neoplastic tissues.

We have demonstrated that Herr's 4 1/2 clearing fluid, developed for use with plant tissues, can be successfully used for the microscopic examination of thick sections of normal and neoplastic mammalian tissues. Rat Novikoff hepatoma, rat liver, and human colon and skin samples were fixed in Bouin's, stained with iron hematoxylin, treated with Herr's 4 1/2 clearing fluid and examined by phase contrast microscopy. Tissue architecture and cytological detail were easily observed by focusing through tissue sections as thick as 70 mu. The method permits rapid microscopic examination of mammalian tissues and enables the investigator to detect readily morphological abnormalities within a tissue.     

Comparison of two methods for linear measurement of ossified tissue in cleared mouse fetuses

Two methods of measuring cranial structures, the direct method and a photographic method, have been compared in day 18 mouse fetuses that have been stained with alizarin red S and cleared. Variation was determined for repeated measurements with each technique of the same specimen as well as the variation among specimens. There was variation in the results of direct measurements of the same specimens, but this was significantly (P less than 0.05) less than the variation among specimens, and the variation of measurements of the same specimen made using the photographic method was not significantly (P greater than 0.2) different from the variation among the specimens, and the variation of the specimens was not significantly (P greater than 0.2) different between the photographic and direct methods. Direct measurement is recommended for making linear measurements on the cleared skull. The photograph method allows better delineation of angles and calculation of surface area.     

Optical clearing methods: An overview of the techniques used for the imaging of 3D spheroids

Spheroids have emerged as in vitro models that reproduce in a great extent the architectural microenvironment found in human tissues. However, the imaging of 3D cell cultures is highly challenging due to its high thickness, which results in a light-scattering phenomenon that limits light penetration. Therefore, several optical clearing methods, widely used in the imaging of animal tissues, have been recently explored to render spheroids with enhanced transparency. These methods are aimed to homogenize the microtissue refractive index (RI) and can be grouped into four different categories, namely (a) simple immersion in an aqueous solution with high RI; (b) delipidation and dehydration followed by RI matching; (c) delipidation and hyperhydration followed by RI matching; and (d) hydrogel embedding followed by delipidation and RI matching. In this review, the main optical clearing methods, their mechanism of action, advantages, and disadvantages are described. Furthermore, the practical examples of the optical clearing methods application for the imaging of 3D spheroids are highlighted.     

An overview of same-sex mounting in turtles and tortoises

Same-sex mounting is an aspect of animal behavior that has received increased attention in recent years in an attempt to improve our limited understanding of the possible causal mechanisms. Here, to our knowledge, we review for the first time same-sex mounting in turtles and tortoises. To this end, we have compiled data on same-sex mounts in 13 chelonian species and discuss the data together with those hypotheses most commonly raised in turtle studies, namely, the intrasexual conflict, maladaptation, mistaken identify, and opposite-sex deprivation hypotheses. Compilation of the data revealed that in almost every species with reports of same-sex mounting there were dominance relationships mediated by aggressive/submissive interactions; most of these cases occurred in captivity, and the sex ratios were not skewed. We discuss future research directions, providing initial ideas of experimental testing on each hypothesis, in the hope of directing more research effort to this field.     

Quantitative assessment of optical clearing methods in various intact mouse organs

Various tissue optical clearing techniques have sprung up for large volume imaging. However, there are few methods showed clearing and imaging data on different organs while most of them were focused on mouse brain, and as a result, it is difficult to select the suitable method for organs in practical applications due to lack of quantitative evaluation and comprehensive comparison. Therefore, it is necessary to evaluate and compare the performances of clearing methods for different organs. In this paper, several typical optical clearing methods were applied, including 3DISCO, uDISCO, SeeDB, FRUIT, CUBIC, ScaleS and PACT to clear intact brain, heart, kidney, liver, spleen, stomach, lung, small intestine, skin and muscle. The clearing efficiency, sample deformation, fluorescence preservation and imaging depth of these methods were quantitatively evaluated. Finally, based on the systemic evaluation of various parameters described above, the appropriate clearing method for specific organ including kidney or intestine was screened out. This paper will provide important references for selection of appropriate clearing methods in related researches.      

一种用于食草动物粪便显微组织分析的临时装片新技术

粪便显微组织分析法是研究食草动物食性的主要方法,其常规装片技术需要使用Hoyer's 装片介质对植物碎片进行封片,而Hoyer's 封片液的粘性易导致植物碎片在装片过程中发生卷曲和重叠,影响植物碎片的识别效果。本文提出的新装片技术采用没有粘性的饱和NaCl 溶液代替Hoyer's 装片介质,结合特定的定量取样方法和装片程序,可以有效地减少植物碎片的卷曲率和重叠率。对比试验显示,新装片技术可使植物碎片卷曲率从10.4% 下降至3.8%,重叠率从25% 下降至8.1% ,说明新装片技术在减少植物碎片卷曲和重叠方面明显优于常规装片方法。     

An easy technique for the clearing of histochemically stained plant tissue

The sites of GUS-activity in strongly pigmented whole-mount preparations or thick handmade sections of plant material can easily be visualized by clearing it with chlorallactophenol (CLP), without affecting the ClBr-indigo precipitate.     

Enantioselective analysis of atenolol in biologic fluids: comparison of liquid-liquid and solid-phase extraction methods

In this study we evaluated a liquid-liquid extraction procedure and a solid-phase extraction procedure for sample preparation for the enantioselective analysis of atenolol in plasma and urine by high-performance liquid chromatography. A Chiralcel OD-H column was used for the resolution of atenolol enantiomers with hexane-ethanol (85:15, v/v) plus 0.1% diethylamine as the mobile phase. In the liquid-liquid extraction procedure, atenolol was extracted from alkalinized body fluids with 5 ml chloroform-2-propanol (4:1, v/v). In the solid-phase extraction procedure, atenolol was isolated from plasma using a C8 column and methanol. Both extraction procedures were efficient in recovering atenolol and removing endogenous interferents. The RSDs and deviation from nominal values were lower than 10% for both within-day and between-day assays. The results show that there were no statistically significant differences in between-day variation. The t-test showed that there were no significant differences between the real concentrations and the determined concentrations. The limit of quantitation was 10 ng/ml and the linear range was 10-5,000 ng/ml for both methods. These methods can be used in pharmacokinetic studies.     

An ultrastructural study of early endosperm development and synergid changes in unfertilized cotton ovules

Excised, unfertilized cotton (Gossypium hirsutum L.) ovules were cultured for 1–5 days postanthesis and embryo-sac development was studied with the electron microscope. In some ovules the two polar nuclei fuse and the diploid endosperm nucleus goes through a limited number of free nuclear divisions after 2–3 days in culture. Each nucleus has two nucleoli, in contrast to nuclei of fertilized triploid endosperm which have three nucleoli. Precocious cell walls form between the endosperm nuclei on the 3rd day in culture. The morphology of the plastids, mitochondria, rough endoplasmic reticulum (RER), dictyosomes and microbodies, and the amount of starch and lipid in the diploid cellular endosperm are similar to those of the central cell. A few large helical polysomes appear close to plastids and mitochondria. After 2 days in culture, one of the two synergids in the unfertilized cultured ovules shows degenerative changes which in fertilized ovules are associated with the presence of the pollen tube, i.e., increase in electron density, collapse of vacuoles, irregular darkening and thickening of mitochondrial and plastid membranes, disappearance of the plasmalemma and the membranes of the plasmalemma and the membranes of the RER. The second synergid remains unchanged in appearance. The egg cell does not shrink or divide or show structural changes characteristic of the cotton zygote. Embryo-sac development is arrested on the 4th and 5th days in culture. The nucellus continues growth and at 14 days crushes the degenerate embryo sac.     

An ultramicro method for quantitation of amino acids in biological fluids

A modification of the commercially available SZ-14 reorienting density gradient rotor is described whereby continuous sample flow with density gradient isopycnic banding may be utilized. This permits the fractionation of large volumes of dilute homogenates with excellent recovery and purity. The technique is demonstrated for the isolation of nuclei and particles of mitochondrial size.     

Pattern diversity analysis of a clearing in a Quercus cerris wood

Linear transects were used to examine the spatial structure of the vegetation in a clearing in a Quercus cerris wood in Central Italy. A grassland-margin-shrubland-woodland gradient was identified.Multivariate classification and ordination methods and pattern diversity analysis were used to detect both floristic variation and spatial organization. One of the transects shows a clear gradient of variation as a function of space, crossing the floristically and structurally most heterogeneous part of the clearing. For the detailed analysis of this transect a sectorization is achieved. Each sector is characterized according to its floristic types and to the internal spatial organization.The pieces of information derived from the different analyses were concordant. In particular, the validity of the floristic types in the qualification of the different forms of spatial organization is confirmed.