New type of needle for obtaining large samples of human adipose tissue

A new type of needle for obtaining human fatty tissue samples is described. With this instrument 350-600 mg of tissue may be obtained less traumatically than with previously described instruments.     

Evaluation of a freeze-clamping technique designed for two- and three-dimensional metabolic studies of rat liver in vivo. Quenching efficiency and effect of clamping on tissue morphology

In order to apply a previously described freeze-clamping technique (B. Quistorff and B. Chance, 1980, Anal. Biochem.108, 237–248) in three-dimensional metabolic studies, it is necessary to clarify to which extent tissue morphology as well as metabolic state is preserved in the part of the freeze-clamped sample used for such studies. The present paper reports a comparative histological examination of rat liver, either freeze-clamped, applying the technique mentioned, or frozen without compression. It is demonstrated that neither the intra- nor the interlobular morphology of the central part of the sample is disturbed by the freeze-clamping process. In clamped as well as unclamped liver samples portal tracts could be identified, i.e., distinguished from efferent veins, in a depth below the capsule of 100–200 μm. Measurements of ATP, ADP, AMP, and P_i in the freeze-clamped sample at increasing distance from the surface indicate that for in situ freezing, significant metabolic changes did not occur until a depth of about 1 mm, while a delay of freezing of about 6 s seems to cause metabolic changes in the entire sample.     

Automated analysis and survival selection of anchorage-dependent cells under normal growth conditions

An instrument is described that can automatically analyze and select for a subpopulation of anchorage-dependent cells in tissue culture. Cells that label with fluorescently tagged antibodies or demonstrate structural variations are saved from exposure to a destructive high-intensity argon laser beam. The surviving population may then be cloned. The cell selection may occur in a tissue culture plate or in a microflow incubator which is designed to maintain a constant flow of media at 37 degrees C across cells growing on a glass coverslip. This incubator sits on an inverted microscope which focuses the laser beam to a diameter as small as 1 micron. A high-speed computer-controlled two-dimensional stage moves the cells past the beam for analysis, the results of which determine the fate of each cell: whether it is to be destroyed by radiant energy or selected for survival and subsequent proliferation. Another selection strategy performed by the instrument involves growing the cells on a thin, blackened polyester film which can be cut by the argon laser beam. Cells selected for cloning are then circumscribed. The heat of cutting welds the circumscribed film to a plastic coverslip surface or tissue culture chamber bottom. Nonselected cells may be removed by pulling the unattached polyester sheet from the attachment surface. The selected cells remain on polyester film disks welded to the plastic. Selections may be done automatically under computer control or manually by operator direction of stage movements. This instrument extends the art of automated cell selection and analysis to normal cell lines that must maintain cell-substratum contact (anchorage dependence) for differentiated cell function, e.g., neurons, fibroblasts, or kidney cells.     

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.     

Modification of the Falck-Hillarp formaldehyde fluorescence method using the vibratome®: simple,rapid and sensitive localization of catecholamines in sections of unfixed or formalin fixed brain tissue

Summary Two modifications of the original Falck-Hillarp formaldehyde fluorescence technique are presented, both based on a recently introduced instrument, the Vibratome^®, which permits cutting of unembedded tissue with a section thickness down to 10 ,.The first modification involves sectioning of unfixed tissue at a temperature below +5°C, subsequent air drying and reaction with formaldehyde vapours. In the second procedure formalin fixed tissue is cut and processed as described above. It is essential that both formalin fixation and cutting of the fixed tissue takes place at a low temperature to avoid diffusion of the catecholamines.The results show that with both techniques central CA neurons can be visualized with a high degree of sensitivity. Furthermore, since the sections are free from fractures—a common problem in freeze-dried tissues—the Vibratome^® technique represents a valuable tool for mapping studies. It may also be added that since many steps of the original procedure are omitted the present techniques are also more rapid and simple. It is pointed out that using the Vibratome^® procedure on formalin fixed tissue, it will be possible to combine e.g. cholinesterase staining or Fink-Heimer silver impregnation or immunofluorescent studies with the Falck-Hillarp technique on serial or even on the same sections.     

Electron microscopy of high pressure frozen samples: bridging the gap between cellular ultrastructure and atomic resolution

Transmission electron microscopy has provided most of what is known about the ultrastructural organization of tissues, cells, and organelles. Due to tremendous advances in crystallography and magnetic resonance imaging, almost any protein can now be modeled at atomic resolution. To fully understand the workings of biological “nanomachines” it is necessary to obtain images of intact macromolecular assemblies in situ. Although the resolution power of electron microscopes is on the atomic scale, in biological samples artifacts introduced by aldehyde fixation, dehydration and staining, but also section thickness reduces it to some nanometers. Cryofixation by high pressure freezing circumvents many of the artifacts since it allows vitrifying biological samples of about 200 μm in thickness and immobilizes complex macromolecular assemblies in their native state in situ. To exploit the perfect structural preservation of frozen hydrated sections, sophisticated instruments are needed, e.g., high voltage electron microscopes equipped with precise goniometers that work at low temperature and digital cameras of high sensitivity and pixel number. With them, it is possible to generate high resolution tomograms, i.e., 3D views of subcellular structures. This review describes theory and applications of the high pressure cryofixation methodology and compares its results with those of conventional procedures. Moreover, recent findings will be discussed showing that molecular models of proteins can be fitted into depicted organellar ultrastructure of images of frozen hydrated sections. High pressure freezing of tissue is the base which may lead to precise models of macromolecular assemblies in situ, and thus to a better understanding of the function of complex cellular structures.     

A rapid and inexpensive method for isolation of total DNA from dehydrated plant tissue

We describe an inexpensive method for dehydration of plant tissue and extraction of high molecular weight DNA. Tissue is dried for 12 to 24 hours in a food dehydrator and subsequently powdered for DNA extraction. Dicot tissue can be powdered in centrifuge tubesen masse using a commercial paint mixer and glass beads. With the use of the paint mixer, tissue never touches common surfaces that might lead to cross contamination, a potential benefit when the DNA is to be used for PCR reactions. The DNA is of a quality equal to that obtained from either lyophilized or fresh frozen tissue (commonly used in many labs). The advantages of the described procedure are that it is fast, does not require expensive equipment (e.g., lyophilizer) and can be used in situations where large numbers of samples must be extracted.     

Preparation-dependent distribution of intramembranous particles in freeze-fracture replicas of the neuromuscular junction of the locust Schistocerca gregaria

Summary Freeze-fracture replicas of the neuromuscular junction were prepared from untreated retractor unguis muscles of the locust Schistocerca gregaria that were rapidly frozen by contact with a copper block cooled by liquid helium. These replicas were compared with others prepared from tissue following fixation with glutaraldehyde and cryoprotection in glycerol. Freeze-fracture of rapidly frozen tissue produced replicas of high quality with little evidence of tissue damage by ice crystals in the superficial layers. The gross fracturing characteristics of the neuromuscular junction were consistent with replicas from fixed and cryoprotected tissue; all of the membrane specializations were recognisable but with some alterations in infrastructure. In tissue replicas prepared using either method intramembranous particles in the presynaptic membrane were arranged in a bar-like array. The intramembranous particles of this presynaptic bar array of the rapidly frozen material were large and found on the E-face of the cleaved membrane. This contrasts with the P-face distribution of the comparable particles in muscles fixed in glutaraldehyde and cryoprotected in glycerol, in which they are also smaller and more numerous. This difference in partitioning between rapidly frozen, and fixed, cryoprotected nerve terminals is not found at cholinergic synapses and thus may reflect functional differences between the two types of junction.Indentations of the nerve-terminal membrane occur in replicas from rapidly frozen muscle as well as fixed and cryoprotected muscle suggesting they are not fixation or glycerol-induced artifacts. It is suggested from their position and size that these indentations are more likely to be part of a membrane retrieval system than exocytotic figures.This work was supported by an S.E.R.C. project grant to I.R.D.     

The aluminium content of breast tissue taken from women with breast cancer

The aetiology of breast cancer is multifactorial. While there are known genetic predispositions to the disease it is probable that environmental factors are also involved. Recent research has demonstrated a regionally specific distribution of aluminium in breast tissue mastectomies while other work has suggested mechanisms whereby breast tissue aluminium might contribute towards the aetiology of breast cancer. We have looked to develop microwave digestion combined with a new form of graphite furnace atomic absorption spectrometry as a precise, accurate and reproducible method for the measurement of aluminium in breast tissue biopsies. We have used this method to test the thesis that there is a regional distribution of aluminium across the breast in women with breast cancer. Microwave digestion of whole breast tissue samples resulted in clear homogenous digests perfectly suitable for the determination of aluminium by graphite furnace atomic absorption spectrometry. The instrument detection limit for the method was 0.48 μg/L. Method blanks were used to estimate background levels of contamination of 14.80 μg/L. The mean concentration of aluminium across all tissues was 0.39 μg Al/g tissue dry wt. There were no statistically significant regionally specific differences in the content of aluminium. We have developed a robust method for the precise and accurate measurement of aluminium in human breast tissue. There are very few such data currently available in the scientific literature and they will add substantially to our understanding of any putative role of aluminium in breast cancer. While we did not observe any statistically significant differences in aluminium content across the breast it has to be emphasised that herein we measured whole breast tissue and not defatted tissue where such a distribution was previously noted. We are very confident that the method developed herein could now be used to provide accurate and reproducible data on the aluminium content in defatted tissue and oil from such tissues and thereby contribute towards our knowledge on aluminium and any role in breast cancer.     

A simple method for fixation and microdissection of frozen fresh tissue sections for molecular cytogenetic analysis of cancers.

Microdissection has been widely used for procuring DNA from specific microscopic regions of formalin fixed, paraffin embedded tissue sections. We have developed a method for fixation and microdissection of frozen fresh biopsy tissue sections. Five micrometer frozen fresh tissue sections were fixed with ethanol and stored at room temperature. Well defined regions from hematoxylin and eosin (H & E) stained or unstained sections were briefly steamed and microdissected using a needle. The dissected tissue was digested with proteinase K and DNA was isolated. Whole genome amplifications were obtained by degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR) from these samples. The reliability of this technique was demonstrated by comparing conventional comparative genomic hybridization (CGH) with DOP-PCR-CGH. The advantages of this method are that frozen fresh sections can be fixed easily and stored for more than 4 years, it is easy to microdissect and pick-up very minute regions (0.1 mm(2)), and it is rapid; microdissection and purification can be accomplished within 3 h. Using DNA from microdissected sections, DOP-PCR-CGH revealed genetic abnormalities more accurately than conventional CGH. Although this novel method was demonstrated using DOP-PCR-CGH, we believe that it will be useful for other genetic analyses of specific small regions and cell populations. We also observed whether storage time, H & E staining and crude DNA extracts affected the quality of amplified DNA. DNA integrity was maintained for at least 49 months in ethanol fixed sections that were stored at room temperature, but DNA was gradually degraded after one month if the ethanol fixed sections had been H & E stained and stored. When crude DNA extracts from H & E stained sections were used, the size of the DOP-PCR product was reduced. Our study suggests that ethanol fixed tissue sections may be stored at room temperature for at least 4 years without DNA degradation, the H & E stains may not affect the quality of amplified DNA, but H & E or other components in the staining process may reduce the size of DOP-PCR product, which is critical for the quality of CGH hybridization.     

Preparation of ultra-thin frozen sections of plant tissues for electron microscopy

Summary A method is described, for the first time, by which ultra-thin frozen sections of plant tissues may be prepared for electron microscopy. Sections of both plant and animal tissues were prepared from either unfixed or fixed tissues, without prior dehydration or infiltration of the tissue with a support medium, and with the aid of a Reichert OmU 2 ultra-microtome with freezing attachment.     

Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells – especially those in tissue – are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.     

A simplified method for mouse blastocyst injection

A simplified method for blastocyst injection is described. Two instruments only are used: one micropipette for holding the blastocyst and another for injection. Both instruments are easily made by cutting a pulled capillary. No bevelling or polishing is needed. The instruments may be used for many successive injections.     

A Comparison of Low-Temperature and Ambient-Temperature SEM for Viewing Nematode Faces

Faces of lesion nematodes Pratylenchus teres (populations RTB and JK) and P. zeae or the bacterivore Distolabrellus veechi were observed on frozen specimens with low-temperature scanning electron microscopy and as chemically fixed, critical-point dried specimens with conventional scanning electron microscopy. Amphidial secretions were preserved in chemically fixed but not cryofixed lesion nematodes. Overhanging liplets of chemically fixed D. veechi may be artifactual because they appeared as variably filled, mostly empty membranes when cryofixed. The diagnostically useful lips of the frozen lesion nematodes exhibited six sectors of variable prominence that were absent in chemically fixed specimens. This variability may be due to different degrees of muscle contraction captured during cryofixation, which occurs in milliseconds. This is the first evidence that rarely observed lip sectors in Pratylenchus may be something other than an artifact of shrinkage.     

Special symposium: fixation and tissue processing models

Abstract

Fixation and processing of tissue to paraffin blocks permit thin (4-5 µm) sections of tissues to be cut. Tissues and their subcellular components and surrounding stroma are visualized by cutting thin sections and staining them histochemically or immunohistochemically and viewing the sections using a bright field microscope. During the last century, anatomists and pathologists have used fixation with 10% neutral buffered formalin (10% NBF) as the fixative of choice. Also, both human and veterinary pathologists have trained to use fixation with 10% NBF, so these professionals are reluctant to change the familiar microscopic appearance of diagnostic tissues by using different fixatives. In addition, the effects of tissue processing on the microscopic appearance of tissue essentially has been ignored in most studies. Archives of paraffin blocks of pathological tissue contain essentially paraffin blocks fixed in 10% NBF. Therefore, if retrospective studies use archival paraffin blocks to correlate the molecular features of diseases with their outcomes, the studies must be based on tissue fixed in 10% NBF. Studies of how fixation in 10% NBF interacts with histochemical and immunohistochemical staining are limited in number and most are based on relatively long fixation times (≥36 h). Currently, fixation times in 10% NBF have been reduced to <24 h. Little is known about fixation in 10% NBF and its interaction with tissue processing for any period of fixation, especially short times. Less is known about how fixation of tissues with 10% NBF interacts with more modern assays using immunohistochemistry, real time quantitative polymerise chain reaction (PCR), and techniques that depend on analysis of proteins extracted from paraffin blocks including multiplex immunoassays or mass spectrometry. In general, multiple antibody–antigen combinations are reported not to work in tissues fixed in 10% NBF, i.e., loss of immunorecognition is nearly complete for such antibody–antigen combinations as Ki67/MIB, estrogen receptor alpha (ERα) and Progesterone receptor (PR), and partial for Bcl-2. Several models have been developed to study the interactions of tissue fixation and immunorecognition, but most have viewed the problem with immunorecognition as completely caused by fixation. Also, some of the models discussed in this special symposium do not predict the effects of fixation on frozen tissues fixed in 10% NBF and not processed to paraffin blocks. This article is a brief review of issues attending the use of 10% NBF combined with tissue processing as an interrelated process to study biomarkers identified by immunohistochemistry.     

Reuse of instruments for minimal invasive surgery--status and prospects]

The development of instruments for minimal invasive surgery (MIS) is moving in the direction of the miniaturization of mechanical components, a combination of multiple functions in a single instrument, and the introduction of new techniques, in particular those reducing bleeding and thermal damage when cutting blood vessels. These tendencies have consequences for the reprocessability of the instruments, usually making reprocessing more difficult. In particular cleaning--the removal of contaminations from tiny lumina, joints, etc., is highly demanding. In addition, proof of successful cleaning is difficult, and no standardised method of doing this in practice is currently available. An overview of the problems associated with the reprocessing of instruments for minimal invasive surgery is given, and a numbers of possible solutions are discussed.     

Keratin antigen retrieval in oral mucosal biopsies using microwave processing

Summary In immunohistochemistry, it is well known that the majority of monoclonal antibodies to keratins work best on fresh frozen tissue specimens, yet in clinical practice most biopsies are routinely fixed in formaldehyde. This seriously limits the range of keratins that can be reliably assessed in retrospective studies (particularly where only rare archival material exists) and where subtle changes during tissue differentiation may be important. Antigen retrieval using exposure to microwave radiation is one technique that has been applied successfully to other tumour markers (e.g., p53). However, few papers have used this method when immunolabelling for keratins, in spite of the widespread use of antikeratin antibodies as markers of differentiation. The effect of keratin antigen retrieval using microwave processing was assessed on a range of oral mucosal biopsies, since the oral cavity displays a wide range of keratins. A panel of six well characterized antibodies was chosen: LP34 (Ck1, 5, 6, 18), LH1 (Ck10), LL025 (Ck16), A53 BA2 (Ck19), AE8 (Ck13), and E3 (Ck17). For each specimen, one piece was stored in liquid nitrogen and another piece fixed in formalin. Tissue sections were cut from each and, using the peroxidase avidin biotin technique, keratin expression was recorded for a frozen section, a dewaxed section, and a microwave-heated dewaxed section. Although overall there was a 25% improvement in identification of keratins after microwaving, some antibodies performed better than others. Given that keratins have been shown to be of value in tumour diagnosis, this study suggests that microwave processing of archival material can be valuable adjunct to such analysis.     

Factors that drive the increasing use of FFPE tissue in basic and translational cancer research

The decision to use 10% neutral buffered formalin fixed, paraffin embedded (FFPE) archival pathology material may be dictated by the cancer research question or analytical technique, or may be governed by national ethical, legal and social implications (ELSI), biobank, and sample availability and access policy. Biobanked samples of common tumors are likely to be available, but not all samples will be annotated with treatment and outcomes data and this may limit their application. Tumors that are rare or very small exist mostly in FFPE pathology archives. Pathology departments worldwide contain millions of FFPE archival samples, but there are challenges to availability. Pathology departments lack resources for retrieving materials for research or for having pathologists select precise areas in paraffin blocks, a critical quality control step. When samples must be sourced from several pathology departments, different fixation and tissue processing approaches create variability in quality. Researchers must decide what sample quality and quality tolerance fit their specific purpose and whether sample enrichment is required. Recent publications report variable success with techniques modified to examine all common species of molecular targets in FFPE samples. Rigorous quality management may be particularly important in sample preparation for next generation sequencing and for optimizing the quality of extracted proteins for proteomics studies. Unpredictable failures, including unpublished ones, likely are related to pre-analytical factors, unstable molecular targets, biological and clinical sampling factors associated with specific tissue types or suboptimal quality management of pathology archives. Reproducible results depend on adherence to pre-analytical phase standards for molecular in vitro diagnostic analyses for DNA, RNA and in particular, extracted proteins. With continuing adaptations of techniques for application to FFPE, the potential to acquire much larger numbers of FFPE samples and the greater convenience of using FFPE in assays for precision medicine, the choice of material in the future will become increasingly biased toward FFPE samples from pathology archives. Recognition that FFPE samples may harbor greater variation in quality than frozen samples for several reasons, including variations in fixation and tissue processing, requires that FFPE results be validated provided a cohort of frozen tissue samples is available.     

Prediction of personalized microRNA activity

MicroRNAs have been known to regulate almost all physiological and pathological processes by suppressing their target genes. In humans, more than 1000 microRNAs have been identified, each of which targets dozens or even hundreds of genes. Facing this huge repertoire of microRNA targeting, it is important to identify which microRNAs are active, i.e., down-regulating their targets, in specific physiological or pathological conditions. Predicting active microRNAs is different from predicting microRNA targets because the authentic target genes of a microRNA are often not directly and solely regulated by that microRNA, leading to inconsistent expression changes between the microRNA and its true targets. Several computational programs have been proposed to predict the activity of a microRNA from the expressions of its target genes. These programs performed well when being applied on the expression data obtained from distinct tissue types or from experiments that transfect a microRNA into cells (i.e., non-physiological). But the performance of microRNA activity prediction is not clear on the expression data from the same tissue type in two physiological conditions, e.g., liver tissues from cancer patients and healthy people. In this work, we evaluate the performance of two microRNA activity prediction programs using seven expression data sets, all of which compare samples in two physiological conditions, as well as propose a new approach that predicts microRNA activity with an accuracy of over 80%. Unlike current methods, which predict active microRNAs by comparing two groups of samples, e.g., tumor versus normal, our new approach compares each diseased sample with all the samples in the control group. In other words, it can predict the microRNA activity of a person. In this work, this new application is named to predict “personalized microRNA activity”.     

Controlling the freezing process: a robotic device for rapidly freezing biological tissues with millisecond time resolution

A robotic cryogenic device was developed which allows freezing of thick biological tissues with millisecond time resolution. The device consists of two horizontally oriented hammers (pre-cooled with liquid N(2)) driven by two linear servo-motors. The tissue sample is bathed in Ringers contained in a chamber which drops rapidly out of the way just as the hammers approach. A third linear motor is vertically oriented, and permits the rapidly dropping chamber to smoothly decelerate. All movements were performed by the three motors and four solenoids controlled by a PC. Mechanical adjustments, that change the size of the gap between the hammers at the end position, permit the final thickness of the frozen tissue to be varied. Here we show that the freezing time increased with the square of the final thickness of the frozen bundle. However, when bundles of different original thicknesses (up to at least 1mm) were compressed to the same final thickness (e.g., 0.2mm), they exhibited nearly equal freezing times. Hence, by being able to adjust the final thickness of the frozen bundles, the device not only speeds the rate of freezing, but standardizes the freezing time for different diameter samples. This permits the use of freezing for accurate determination of the kinetics of cellular processes in biological tissue.