A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed,paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

A comparison of microwave heating and proteolytic pretreatment antigen retrieval techniques in formalin fixed, paraffin embedded tissues

Antigen retrieval (AR) is a technique that re-exposes epitopes in formalin fixed, paraffin embedded sections and makes them detectable by immunohistochemistry. We compared the effects of two AR procedures, enzyme digestion and microwave heating, on immunostaining of vimentin and desmin in formalin fixed, paraffin embedded tissues. Our results showed that AR is necessary for vimentin and desmin immunostaining in tissues fixed in formalin for more than 48 h. With prolonged fixation times, microwave heating showed better results than enzyme digestion for AR. The same results were obtained using 1% zinc sulfate or Citra Plus solution as retrieval solutions for microwave heating. We recommend microwave heating for AR, because it is easier to use and produces better results compared to enzyme treatment.     

Comparison of fine needle aspiration biopsy and paraffin embedded tissue sections for measuring AgNOR proteins

Abstract

Paraffin embedded tissue sections and fine needle aspiration biopsy (FNAB) are important methods for diagnosis. We compared thyroid tissue obtained by FNAB to paraffin embedded sections to determine whether there were differences in detection of the amounts of argyrophilic nucleolar organizing region (AgNOR) proteins. Twenty-two patients with papillary thyroid carcinoma were included in the study. Slides were prepared with both FNAB tissue and 3 μm sections of paraffin embedded tissue, and stained for AgNOR. One hundred nuclei per individual were evaluated; total AgNOR number/nucleus (TAn/TNn) and total AgNOR area/nuclear area (TAa/TNa) of individual cells were determined. Mean TAn/TNn and TAa/TNa values were 4.800 ± 1.118 and 13.382 ± 2.612, respectively, for FNAB samples; corresponding values were 2.406 ± 0.649 and 8.49 ± 0.893, respectively, for paraffin embedded sections. The differences between FNAB materials and paraffin embedded tissue sections were significant for the mean TAn/TNn and TAa/TNa values. Significant differences in the amounts of AgNOR protein detected were found between FNAB and paraffin embedded tissue sections.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

Fluorescence in situ hybridization in paraffin tissue sections: pretreatment protocol

Fluorescence in situ hybridization has found wide application in the enumeration of gene and chromosome copy number both in isolated cells and in tissue sections. However, the technique has been less widely used than would be expected in formalin-fixed paraffin processed (archival) tissue. This article describes a method for assessing archival tissue sections, following pretreatment, before applying DNA probes, that gives consistent, reliable results.     

Purification of DNA from formaldehyde fixed and paraffin embedded human tissue

The ability to isolate DNA from preserved human tissues would provide numerous experimental opportunities. In this report it is shown that DNA can be extracted from tissues prepared for routine histopathological examination (i.e., fixed with formaldehyde and embedded in paraffin). Although the extracted DNA is not intact, it is double stranded, cleavable with restriction endonucleases, and suitable for a variety of standard techniques used in molecular biology.     

DNA cytofluorometry in micro-dissected paraffin embedded breast tissue: description of a method

DNA microfluorometry on smears obtained from paraffin embedded tissue has been shown to be a distinctive possibility. In this paper a simple method for the detachment of cells from mammary ducts and ductules is described. Areas of interest were selected in conventional slides stained with Haematoxylin and Eosin. The corresponding paraffin embedded block was then dewaxed. The areas under study were retraced with a stereomicroscope and the cells within ducts and ductules were scraped out with a 0.4 mm diameter fine needle. Cells were isolated with mechanical and enzymatic procedures and stained with the Feulgen reaction. The DNA content of single cells was then measured using a microfluorometer.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8–12 h at –50 to –40°C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45°C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4°C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

脂肪组织蛋白质组学研究进展

脂肪组织不仅是机体的能量储存库,而且也是重要的内分泌器官。脂肪组织分泌多种激素和细胞因子,参与调节机体多种生理和病理过程。目前飞速发展的蛋白质组学技术,为深入研究脂肪发育的分子机制及其代谢紊乱发生的遗传机理提供了有力的工具。对蛋白质组学在脂肪组织中的研究进展进行了综述,为脂肪组织的发育调控及代谢疾病的治疗提供了新的思路。     

Isolation of high quality protein samples from punches of formalin fixed and paraffin embedded tissue blocks

In general, it is believed that the extraction of proteins from formalin-fixed paraffin embedded samples is not feasible. However, recently a new technique was developed, presenting the extraction of non-degraded, full length proteins from formalin fixed tissues, usable for western blotting and protein arrays. In the study presented here, we applied this technique to punch biopsies of formalin fixed tissues embedded in paraffin to reduce heterogeneity of the tissue represented in sections, and to ensure analysing mainly defined cellular material. Successful extraction was achieved even from very small samples (0.7 mm(3)). Additionally, we were able to detect highly glycosylated proteins and protein modification, such as phosphorylation. Interestingly, with this technique it is feasible to extract high quality proteins from 14 year old samples. In summary, the new technique makes a great pool of material now usable for molecular analysis with high throughput tools.     

Modulation of white adipose tissue proteome by aging and calorie restriction

Aging is associated with an accrual of body fat, progressive development of insulin resistance and other obesity comorbidities that contribute to decrease life span. Caloric restriction (CR), which primarily affects energy stores in adipose tissue, is known to extend life span and retard the aging process in animal models. In this study, a proteomic approach combining 2‐DE and MS was used to identify proteins modulated by aging and CR in rat white adipose tissue proteome. Proteomic analysis revealed 133 differentially expressed spots, 57 of which were unambiguously identified by MS. Although CR opposed part of the age‐associated protein expression patterns, many effects of CR were on proteins unaltered by age, suggesting that the effects of CR on adipose tissue are only weakly related to those of aging. Particularly, CR and aging altered glucose, intermediate and lipid metabolism, with CR enhancing the expression of enzymes involved in oxalacetate and NADPH production, lipid biosynthesis and lipolysis. Consistently, insulin‐β and β3‐adrenergic receptors were also increased by CR, which denotes improved sensitivity to lipogenic/lipolytic stimuli. Other beneficial outcomes of CR were an improvement in oxidative stress, preventing the age‐associated decrease in several antioxidant enzymes. Proteins involved in cytoskeleton, iron storage, energy metabolism and several proteins with novel or unknown functions in adipose tissue were also modulated by age and/or CR. Such orchestrated changes in expression of multiple proteins provide insights into the mechanism underlying CR effects, ultimately allowing the discovery of new markers of aging and targets for the development of CR‐mimetics.     

Transcriptional profiling of formalin fixed paraffin embedded tissue: pitfalls and recommendations for identifying biologically relevant changes

Expression profiling techniques have been used to study the biology of many types of cancer but have been limited to some extent by the requirement for collection of fresh tissue. In contrast, formalin fixed paraffin embedded (FFPE) samples are widely available and represent a vast resource of potential material. The techniques used to handle the degraded and modified RNA from these samples are relatively new and all the pitfalls and limitations of this material for whole genome expression profiling are not yet clarified. Here, we analyzed 70 FFPE tongue carcinoma samples and 17 controls using the whole genome DASL array covering nearly 21000 genes. We identified that sample age is related to quality of extracted RNA and that sample quality influences apparent expression levels in a non-random manner related to gene probe sequence, leading to spurious results. However, by removing sub-standard samples and analysing only those 28 cancers and 15 controls that had similar quality we were able to generate a list of 934 genes significantly altered in tongue cancer compared to control samples of tongue. This list contained previously identified changes and was enriched for genes involved in many cancer-related processes such as tissue remodelling, inflammation, differentiation and apoptosis. Four novel genes of potential importance in tongue cancer development and maintenance, SH3BGL2, SLC2A6, SLC16A3 and CXCL10, were independently confirmed, validating our data. Hence, gene expression profiling can be performed usefully on archival material if appropriate quality assurance steps are taken to ensure sample consistency and we present some recommendations for the use of FFPE material based on our findings.     

Optimal molecular profiling of tissue and tissue components

Isolation of well-preserved pure cell populations is a prerequisite for sound studies of the molecular basis of any tissue-based biological phenomenon. This article reviews current methods for obtaining anatomically specific signals from molecules isolated from tissues, a basic requirement for productive linking of phenotype and genotype. The quality of samples isolated from tissue and used for molecular analysis is often glossed over or omitted from publications, making interpretation and replication of data difficult or impossible. Fortunately, recently developed techniques allow life scientists to better document and control the quality of samples used for a given assay, creating a foundation for improvement in this area. Tissue processing for molecular studies usually involves some or all of the following steps: tissue collection, gross dissection/identification, fixation, processing/embedding, storage/archiving, sectioning, staining, microdissection/annotation, and pure analyte labeling/identification and quantification. We provide a detailed comparison of some current tissue microdissection technologies, and provide detailed example protocols for tissue component handling upstream and downstream from microdissection. We also discuss some of the physical and chemical issues related to optimal tissue processing, and include methods specific to cytology specimens. We encourage each laboratory to use these as a starting point for optimization of their overall process of moving from collected tissue to high quality, appropriately anatomically tagged scientific results. In optimized protocols is a source of inefficiency in current life science research. Improvement in this area will significantly increase life science quality and productivity. The article is divided into introduction, materials, protocols, and notes sections. Because many protocols are covered in each of these sections, information relating to a single protocol is not contiguous. To get the greatest benefit from this article, readers are advised to read through the entire article first, identify protocols appropriate to their laboratory for each step in their workflow, and then reread entries in each section pertaining to each of these single protocols.     

A silver carbonate method for cell counts of neurons and glial elements on paraffin embedded brain tissue.

A modification of the Del Rio-Hortega method for the demonstration of central nervous system elements is presented. This silver impregnation technique is particularly useful for the classification of cell types for quantitative differential cell counts. Formalin fixed paraffin sections are immersed in formol-ammonium bromide for 1 1/2 hours; this solution is an excellent mordant for various silver nitrate stains. The samples are stained for 20 to 60 minutes in a silver carbonate solution (25 ml of 25% silver nitrate combined with 200 ml of 5% sodium carbonate) and then reduced in a 1% formaldehyde solution to which 20 drops of acetic acid have been added. Finally, the slides are fixed in sodium thiosulfate, rinsed in tap water, dehydrated, cleared, and mounted. This procedure will enable this investigator to identify neurons, oligodendroglia, and astrocytes on the basis of their nuclear staining as well as to demonstrate the laminae of brain tissue since the method allows differentiation of cell layers and fiber tracts.     

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using ''filter aided sample preparation'' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on ''pipette-tip'' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.