Micropreparation of single secretory glands from the carnivorous plant Nepenthes

A rapid mechanical micropreparation technique has been developed to isolate multicellular glands, here from Nepenthes pitchers, based on a microdissection platform. The method is an alternative to laser capture dissection because fresh plant tissue can be used directly without previous fixation. Subsequent experiments, such as polymerase chain reaction (PCR)-based detection of an individual gene encoding a thaumatin-like protein and RNA extraction for gene expression analysis, have been successfully added to prove the quality of the prepared biological material. The procedure described is adaptable to a broad range of plant species and should find wide application in the preparation of multicellular glands or other tissues.     

A procedure for the purification of ferritin from human liver by heating a methanol-treated homogenate

A simple, rapid technique for purification of ferritin from human liver tissue is described. Methanol, at a final concentration of 40% (v/v) in liver homogenate, precipitates the majority of proteins but does not affect ferritin. Subsequent heating of this homogenate at 75 degrees C for 10 min results in a purified ferritin preparation as judged by immunoelectrophoresis and polyacrylamide gel electrophoresis. The resultant purified ferritin contained the same amount of iron as the original endogenous ferritin. There were no significant differences (paired t tests) in the amount of protein in the purified ferritin preparation when measured by rocket immunoelectrophoresis and by the Lowry procedure, suggesting that the antigenecity of ferritin was unaffected by the methanol and heat treatment. Both endogenous liver ferritin and radiolabeled human liver ferritin added to liver homogenates were recovered after methanol and heat treatment with similar yields (77 +/- 7% and 70 +/- 2%, respectively) when compared with the standard treatment of heating a homogenate at 75 degrees C. The overall ferritin yield with this rapid procedure was 40%.     

The Photography of Ice Formation in Plant Tissue

A procedure is described which enables ice to be photographedas it forms at an exposed cut surface of plant tissue. Epi-illuminationwith u.v. light is used to excite the fluorescence of substancesapplied to the cut surface in aqueous solution. The fluoresceris chosen to have a high quantum yield when absorbed on plantcell walls and when trapped in ice crystals. Time lapse photographyusing this method shows that the first tissue to freeze in ahardy plant is the vascular system.     

Tissue microdissection techniques in quantitative genome and gene expression analyses

Current advances in quantitative genome and gene expression analyses allow precise molecular genetic fingerprinting of tumor tissues. A crucial factor for the reliability of the data obtained with these refined techniques is the use of morphologically well-defined cell populations. Microdissection technology has been developed to procure pure cell populations from specific areas of tissue sections under microscopic control. This review covers techniques of tissue microdissection in the context of commonly used methods of quantitative genome and gene expression analysis. The first part of the review will summarize the technical aspects of various methods developed for tissue microdissection. In the latter part, current applications of quantitative genome and gene expression analysis techniques employed in microdissected tissue samples will be described.     

Fiftyfold amplification of the Lowry protein assay

The blue product of the Lowry et al. (1951, J. Biol. Chem. 193, 265-275) reaction interacts with malachite green (MG), inducing a change in the visible light spectrum. At A690 nm the absorbance of malachite green solutions increases 10-fold in the presence of Lowry blue (LB). Under the optimum conditions, 0.01 A700 nm unit of Lowry blue produces a change in A690 nm unit of malachite green of 0.5 and the delta A690 nm is a linear function of Lowry blue concentration. Conditions under which this 50-fold amplification can be exploited to detect less than 100 ng of protein (or 4 micrograms X ml-1) are described. A number of chemicals including sodium dodecyl sulfate can interfere with the assay but a strategy has been devised to overcome these problems. Amplification of the Lowry assay appears to involve a cooperative interaction between malachite green and the Lowry blue product such that about 23 molecules of malachite green undergo a spectral shift per molecule of a model reactant such as tyrosine. Malachite green can be used to amplify the molybdenum blue signal obtained in other assays. Less than 10 pmol of tyrosine can be detected using this procedure. Lowry blue also interacts with auramine O, giving a large increase in A500 nm and a 40-fold amplification of the LB signal. As with malachite green, there is a cooperative interaction between auramine O and LB. About 72 molecules of auramine O undergo a spectral shift per molecule of tyrosine. The product of this reaction is also fluorescent and could be exploited in a protein assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimizing Frozen Sample Preparation for Laser Microdissection: Assessment of CryoJane Tape-Transfer System?

Laser microdissection is an invaluable tool in medical research that facilitates collecting specific cell populations for molecular analysis. Diversity of research targets (e.g., cancerous and precancerous lesions in clinical and animal research, cell pellets, rodent embryos, etc.) and varied scientific objectives, however, present challenges toward establishing standard laser microdissection protocols. Sample preparation is crucial for quality RNA, DNA and protein retrieval, where it often determines the feasibility of a laser microdissection project. The majority of microdissection studies in clinical and animal model research are conducted on frozen tissues containing native nucleic acids, unmodified by fixation. However, the variable morphological quality of frozen sections from tissues containing fat, collagen or delicate cell structures can limit or prevent successful harvest of the desired cell population via laser dissection. The CryoJane Tape-Transfer System®, a commercial device that improves cryosectioning outcomes on glass slides has been reported superior for slide preparation and isolation of high quality osteocyte RNA (frozen bone) during laser dissection. Considering the reported advantages of CryoJane for laser dissection on glass slides, we asked whether the system could also work with the plastic membrane slides used by UV laser based microdissection instruments, as these are better suited for collection of larger target areas. In an attempt to optimize laser microdissection slide preparation for tissues of different RNA stability and cryosectioning difficulty, we evaluated the CryoJane system for use with both glass (laser capture microdissection) and membrane (laser cutting microdissection) slides. We have established a sample preparation protocol for glass and membrane slides including manual coating of membrane slides with CryoJane solutions, cryosectioning, slide staining and dissection procedure, lysis and RNA extraction that facilitated efficient dissection and high quality RNA retrieval from CryoJane preparations. CryoJane technology therefore has the potential to facilitate standardization of laser microdissection slide preparation from frozen tissues.     

改良手工显微切割法获取特定细胞提取高质量RNA

探讨显微切割过程中有效保持RNA完整性的组织固定方法,建立一种简易的手工显微切割法.应用自制“T形板”辅助冰冻切片,100％无水乙醇一次性脱水固定,“排除切割法”获取目的细胞,用TRIzol提取RNA,琼脂糖凝胶电泳和RT-PCR分析RNA质量.“一步法”固定可长时间保存RNA的完整性;从食管癌标本5个特定阶段的细胞中提取的RNA,经电泳和RT-PCR分析均具有较高的质量.无水乙醇“一步法”固定,在显微切割的过程中可有效保持RNA的完整性;T形板和“排除切割法”简化了手工显微切割的操作,提取的RNA质、量均可满足后续分子水平研究的需要.     

Microdissection of the mouse egg

A rapid and efficient method for microdissection of the mouse egg is described. The dissection is carried out in hanging drops of medium surrounded by heavy liquid paraffin oil at room temperature. Eggs are first deformed into a cylindrical shape and then dissected at a predetermined site with a glass needle on a Leitz micromanipulator. The survival rate of the dissected fragments is 75–90% and between 20 and 30 eggs can be dissected in an hour. Development of the dissected eggs is at least as good as that described after other types of manipulation. Cytoplasts and karyoplasts of various sizes can be prepared, as well as gynogenetic and androgenetic eggs with different amounts of cytoplasm. This procedure may help to examine nuclear-cytoplasmic interactions in eggs reconstituted from a variety of fragments.     

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.     

A Preservation Method That Allows Recovery of Intact RNA from Tissues Dissected by Laser Capture Microdissection

We report a novel method for preparing samples for laser capture microdissection. The procedure described here permits extraction of intact RNA while preserving morphology, thus being suitable both for identification of specific cells and for analysis of their gene expression. The method is applicable to both mouse embryos and human tumors and may improve the preparation of cDNA libraries from specific cell types without interfering with histological diagnosis.     

Sensitive isocratic liquid chromatographic assay for the determination of 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin in plasma and tissue with electrochemical detection

A simple extraction procedure and a sensitive high-performance liquid chromatographic (HPLC) method are described for the determination of the photodynamic therapeutic agent 5, 10, 15, 20-tetra(m-hydroxyphenyl)chlorin (mTHPC) in plasma and tumour tissue. Reversed-phase high-performance liquid chromatography was performed on a C₁₈ column (70×4.6 mm I.D.) with a mobile phase of 0.01 M potassium dihydrogenphosphate buffer, pH 2.5-acetonitrile (55:45, v/v) and a coulometric detection (+0.80 V). The mean recoveries of mTHPC in the concentration ranges (5–2000 and 10–1000 ng/ml) were 90 and 89% for plasma and tumour samples, respectively. The procedure for plasma and tissue preparation involved solvent precipitation using methanol combined with ammonia solution and dimethyl sulphoxide (4, 0.2, 0.1, v/v/v) and (2, 0.1, 0.1, v/v/v) for plasma and tissue, respectively. For mTHPC at concentrations ranging from 5 to 2000 ng/ml, the within-day relative standard deviations, based on triplicate determinations were less than 8% and the between-day relative standard deviations calculated by performing extraction procedure of plasma samples on three different days ranged from 3 to 18%. This highly sensitive method, 5 and 10 ng/ml for plasma and tissue respectively, was applied successfully to the determination of mTHPC in mouse tumours for pharmacokinetic studies.     

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.     

Effect of pulsed CO2-laser irradiation on bone tissue

Different dynamic effects on biological tissue caused by pulsed laser radiation are described. It is shown that the parameters of these effects which take place on the bone tissue affected by pulsed CO2-laser radiation are directly dependent on the parameters of these pulses and may be predicted for any concrete application.     

Determination of fumagillin in muscle tissue of rainbow trout using automated ion-pairing liquid chromatography

A high-performance liquid chromatographic assay is described as a routine analytical method for the determination of fumagillin in rainbow trout muscle tissue. Muscle tissue samples (1 g) containing fumagillin were deproteinized with 8 ml of an acetonitrile-water mixture (2:6, v/v). The extracts were purified with a Bond Elut Octyl C₈ cartridge column, washed with a water-methanol mixture (95:5, v/v; 4 ml) and fumagillin was eluted with acetonitrile (1 ml). Analytical separations were performed by reversed-phase HPLC with UV detection at 351 nm under gradient conditions. The mobile phase was acetonitrile-0.005 M tetrabutyl ammonium phosphate in water (pH 7.8). The assay is specific and reproducible within the fumagillin range of 20–1000 ng/g and recovery at 20 ng/g was 69.2%. Sample preparation involves the use of a robotic sample preparation system. Gravimetric validation of all operations enabled Good Laboratory Practices to be observed.     

Protease and ribonuclease activities in bovine pituitary lobes

1. Acid and alkaline protease activities in bovine anterior and posterior pituitary lobes were reinvestigated by measurement of u.v. and Folin-Ciocalteu colour values of trichloroacetic acid-soluble digestion products of denatured haemoglobin. 2. Both lobes of the pituitary gland contain a cathepsin with a pH optimum at 3.8. 3. When release of u.v.-absorbing material was used as the assay there was also an optimum at pH8.3-9.7, but this proved to be due to the release of nucleosides from an endogenous substrate. 4. The presence of a ;cyclizing' ribonuclease active at alkaline pH on endogenous RNA was confirmed by the inhibitory effects of phosphate, arsenate and bentonite. The activity was unaffected by heat, EDTA or metal ions. The enzyme also acted on exogenous RNA. 5. A purified preparation of neurosecretory granules from fresh bovine posterior pituitary lobes was free from alkaline ribonuclease activity. Most of the activity present in the tissue was recovered in the supernatant plus microsomal material. 6. The distribution of RNA did not follow that of the alkaline ribonuclease.     

Interference by Tris buffer in the estimation of protein by the Lowry procedure

Tris buffers were found to distort the measurement of protein by the Lowry method both by decreasing chromophore development with protein and by contributing blank color. Tris at an assay concentration of 0.37 mm markedly affects measured results. Similar Tris effects were observed at all wavelengths between 450 and 800 nm and with diverse protein samples. The distortion due to Tris is not correctable by simple blank correction, but it can be overcome by incorporating the same amount of Tris in the standards used. The distortion at Tris concentrations <0.15 mm appears to be within tolerable limits. No interference or distortion was observed with sodium phosphate buffer to an assay concentration of 40 mm. An automated Lowry procedure is also presented which gives excellent correlation with the manual method and an average coefficient of variation of <4%.