Fluorescence in situ hybridization using an old world monkey Y chromosome specific probe combined with immunofluorescence staining on rhesus monkey tissues.

To date, there is no commercially available Y chromosome probe that can be used for fluorescence in situ hybridization (FISH) for the male rhesus monkey. We have recently generated a probe for FISH with high specificity to the short arm of the rhesus monkey Y chromosome. In this study, we further describe a method that keeps the integrity of tissue-specific antigenic structures for immunofluorescence staining subsequent to FISH on paraffin-embedded rhesus monkey tissues. We have examined this technique in combination with an epithelial cell-specific marker, cytokeratin 8/18 (CK8/18), on various tissues, including jejunum, liver, kidney, and pancreas. CK8/18 and Y chromosome signals were distinctly seen simultaneously on epithelial cells from the same tissue section from male but not female monkeys. These studies indicate that our FISH immunofluorescence technique can be reliably used to identify and phenotype male cells in paraffin-embedded rhesus monkey tissues.     

Co-localization of P2Y1 receptor and NTPDase1/CD39 within caveolae in human placenta

Nucleoside triphosphate diphosphohydrolase-1 (NTPDase1/CD39) is the dominant ecto-nucleotidase of vascular and placental trophoblastic tissues and appears to modulate the functional expression of type-2 purinergic (P2) G-protein coupled receptors (GPCRs). Hence, this ectoenzyme could regulate nucleotide-mediated signalling events in placental tissue. This immunohistochemical and immuno-electron microscopic study demonstrates the expression of NTPDase1/CD39, P2Y1 and P2Y2 receptors in different cell types of human placenta. Specifically P2Y1 has an exclusive vascular distribution whereas P2Y2 is localized on trophoblastic villi. Co-localization of P2Y1 and NTPDase1/CD39 are observed in caveolae, membrane microdomains of endothelial cells. The differential localization of these P2 receptors might indicate their unique roles in the regulation of extracellular nucleotide concentrations in human placental tissues and consequent effects on vascular tone and blood fluidity.     

Application of the peroxidase-antiperoxidase technique for the detection of enterotoxigenic Escherichia coli K99 pilus antigen in paraffin-embedded tissues

The peroxidase-antiperoxidase (PAP) technique was evaluated as a method for detecting enterotoxigenic Escherichia coli (ETEC) K99 pilus antigen in paraffin-embedded tissues of swine. Antigenic reactivity was optimal when tissues were processed in cold methanol according to a modified method of Sainte-Marie. This method of processing adequately preserved tissue morphology. The PAP technique was applicable to tissues where endogenous peroxidase activity was minimal.     

Evaluation of the suitability of ex vivo handled ovarian tissues for optical diagnosis by Raman microspectroscopy

A pilot Raman microspectroscopy study of formalin-fixed, paraffin-embedded, and deparaffinized sections from the same ovarian normal and malignant tissues was carried out. This approach was considered in order to evaluate the suitability of these ex vivo tissue handling procedures in discrimination as well as biochemical characterization. The spectra of formalin-fixed normal and malignant tissues exhibited no contamination due to formalin, which is indicated by the absence of strong formalin peaks; spectral features also show significant differences for normal and malignant tissues. The differences between spectral profiles of deparaffinized normal and malignant tissues are subtle and spectra show few residual sharp peaks of paraffin. Complete dominance of paraffin swamping signals from tissues was observed in the spectra of paraffin-embedded tissues. Principal components analysis (PCA), which was employed for discrimination of tissue type, provided good discrimination for formalin-fixed and paraffin-embedded tissue spectra. PCA of deparaffinized tissues resulted in a poor classification with significant overlap among the clusters. Thus, this study indicates that formalin fixation is the most suitable among the three procedures employed in the study. Significant differences between spectral profiles of normal and malignant formalin-fixed tissues can not only be exploited for discrimination but can also provide information on biochemical characteristics of the tissues. Deparaffinized tissues provide poor discrimination and information on tissue biochemistry is lost. Paraffin-embedded tissues may provide good discrimination, but predominance of paraffin in the spectra could jeopardize biochemical characterization. Prospectively, as a result of the better availability of paraffin-embedded tissues and problems associated with frozen sectioning of formalin-fixed tissues, the results of this study using paraffin-embedded tissues are very encouraging.     

Microwave oven-based technique for immunofluorescent staining of paraffin-embedded tissues

Immunohistochemical analysis of formalin-fixed paraffin-embedded tissues can be challenging due to potential modifications of protein structure by exposure to formalin. Heat-induced antigen retrieval techniques can reverse reactions between formalin and proteins that block antibody recognition. Interactions between antibodies and antigens are further enhanced by microwave irradiation, which has simplified immunohistochemical staining protocols. In this report, we modify a technique for antigen retrieval and immunofluorescent staining of formalin-fixed paraffin-embedded tissues by showing that it works well with several antibodies and buffers. This microwave-assisted method for antigen retrieval and immunofluorescent staining eliminates the need for blocking reagents and extended washes, which greatly simplifies the protocol allowing one to complete the analysis in less than 3 h.     

PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep

A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep. When pure cultures of Cl. perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers). When used to identify Cl. perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl. perfringens. No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.     

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.     

Flow cytometric analysis of DNA in cells obtained from deparaffinized formalin-fixed lymphoid tissues

Flow cytometric analysis of DNA content was performed on nuclear suspensions prepared from fresh and from paraffin-embedded, formalin-fixed lymphoid tissues. We confirmed previous reports that it is possible to obtain nuclear suspensions from deparaffinized, formalin-fixed tissues, suitable for DNA analysis by flow cytometry. We observed a tendency for a larger coefficient of variation (CV) of the DNA measurements in the fixed tissues than in the unfixed material causing abnormalities in 2 of 19 lymphomas to become undetectable. Furthermore, samples from different paraffin blocks of a single tumor with an extra G1 (hyperdiploid) peak showed marked differences in the CV of the hyperdiploid peak while the CV of the diploid peak was similar in all samples. In both benign and malignant lymphoid tissues, the S-phase fraction was higher in paraffin-embedded tissues than in unfixed cells. This difference could be attributed to 4', 6'-diamidino-2-phenylindole dihydrochloride (DAPI), a DNA-binding dye commonly used in this technique. Nevertheless, intermediate and high grade lymphomas from paraffin-embedded tissues generally showed a greater S-fraction than low grade lymphomas, a similar observation as with unfixed tissues. Therefore, DNA content analysis of nuclei extracted from paraffin sections may be inadequate to resolve slight aneuploidy, but the measurement of S-fraction size may remain diagnostically or prognostically valuable. Large retrospective studies will be necessary to determine the clinical impact of this technique in the analysis of lymphomas.     

PCR and in situ hybridization for the detection and localization of a new pathogen Francisella-like bacterium (FLB) in ornamental cichlids

Archived formalin-fixed, paraffin-embedded tissues from 28 diseased ornamental cichlid fish associated with visceral granulomas were examined by polymerase chain reaction (PCR) and in situ hybridization (ISH) for detection of Francisella-like bacteria (FLB). The 16S rDNA FLB-specific primer pair 180f/465r was used on naturally infected ornamental cichlids, resulting in 11 positive cases (39%). Using DNA probes, all 28 cases (100%) showed a positive reaction, and most labeled cells were observed in the visceral granulomas of infected individuals. FLB was detected in cells morphologically resembling epithelioid and endothelioid macrophages. ISH was more sensitive than PCR or routine histopathological examination, based on the examination of archived formalin-fixed, paraffin-embedded tissues in this study. Furthermore, this technique located a new fish pathogen, FLB, in ornamental cichlids. The causative agent was similar to the pathogen inducing systemic granulomas in tilapia.     

PCR detection of Actinobacillus pleuropneumoniaeapxIV gene in formalin-fixed,paraffin-embedded lung tissues and comparison with in situ hybridization

AIMS: Formalin-fixed, paraffin-embedded lung tissues from pigs experimentally infected with 12 Actinobacillus pleuropneumoniae serotypes were used to develop nested PCR for the detection of apxIV gene. METHODS AND RESULTS: The PCR results from formalin-fixed, paraffin-embedded tissues were compared with in situ hybridization. The apxIV gene was detected in formalin-fixed, paraffin-embedded lung tissues from all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes by nested PCR. In situ hybridization produced a distinct positive signal in all 39 pigs experimentally infected with 12 A. pleuropneumoniae serotypes. Agreement rates between nested PCR and in situ hybridization were 100% for the detection of apxIV gene in formalin-fixed paraffin-embedded lung tissues. Acceptable PCR signals were detected from lung tissues fixed for periods up to 180 days. CONCLUSIONS: The apxIV gene is species-specific rather than serotype-specific and is therefore an important diagnostic marker. The nested PCR assay would be a useful method for the detection of apxIV gene to diagnose A. pleuropneumoniae infection when formalin-fixed tissues are submitted. SIGNIFICANCE AND IMPACT OF THE STUDY: This study confirmed the possibility of using formalin-fixed, paraffin-embedded tissues for the diagnosis of A. pleuropneumoniae infection in pigs.     

DNA extraction from archival formalin-fixed, paraffin-embedded tissue sections based on the antigen retrieval principle: heating under the influence of pH.

During the course of diagnostic surgical pathology, pathologists have established a large collection of formalin-fixed, paraffin-embedded tissues that form invaluable resources for translational studies of cancer and a variety of other diseases. Accessibility of macromolecules in the fixed tissue specimens is a critical issue as exemplified by heat-induced antigen retrieval (AR) immunohistochemical (IHC) staining. On the basis of observations that heating may also enhance in situ hybridization (ISH) and the similarity of formalin-induced chemical modifications that occur in protein and in DNA, we designed a study to examine the efficiency of DNA extraction from archival formalin-fixed, paraffin-embedded tissues using an adaptation of the basic principles of the AR technique, i.e., heating the tissue under the influence of different pH values. Archival paraffin blocks of lymph nodes, tonsil, and colon were randomly selected. Each paraffin block was prepared in 34 microtubes. For each paraffin block, one tube was used as a control sample, using a non-heating DNA extraction protocol. The other 33 tubes were tested using a heating protocol under 11 variable pH values (pH 2 to 12) under three different heating conditions (80, 100, and 120C). Evaluation of the results of DNA extraction was carried out by measuring yields by photometry and PCR amplification, as well as kinetic thermocycling (KTC)-PCR methods. In general, lower pH (acid) solutions gave inferior results to solutions at higher pH (alkaline). Heating tissues at a higher temperature and at pH 6-9 gave higher yields of DNA. There appeared to be a peak in terms of highest efficiency of extracted DNA at around pH 9. The average ratios 260:280 of extracted DNA also showed better values for samples heated at 120C. PCR products of three primers showed satisfactory results for DNA extracted from archival paraffin-embedded tissues by heating protocols at pH 6-12, with results that were comparable to the control sample subjected to the standard non-heating, enzymatic DNA extraction method. This study is the first to document the use of heating at an alkaline pH for DNA extraction from archival formalin-fixed, paraffin-embedded tissues, a recommendation based on the principles of AR for protein IHC. These findings may lead to a more effective protocol for DNA extraction from archival paraffin-embedded tissues and may also provide enhanced understanding of changes that occur during formalin-induced modification of nucleic acids.     

A novel procedure for protein extraction from formalin-fixed paraffin-embedded tissues

Most of the archived pathological specimens in hospitals are kept as formalin-fixed paraffin-embedded tissues (FFPE) for long-term preservation. Up to now, these samples are only used for immunohistochemistry in a clinical routine as it is difficult to recover intact protein from these FFPE tissues. Here, we report a novel, short time-consuming and cost-effective method to extract full-length, non-degraded proteins from FFPE tissues. This procedure is combined with an effective and non-toxic deparaffinisation process and an extraction method based on antigen-retrieval, high concentration of SDS and high temperature. We have obtained enough intact protein to be detected by Western blotting analysis. This technique will allow utilising these stored FFPE tissues in several applications for protein analysis helping to advance the translational studies in cancer and other diseases.     

Extraction of cells from paraffin-embedded tissue sections for single-cell DNA cytophotometry

A simple technique is presented for the isolation of cells from paraffin-embedded tissues for Feulgen DNA cytophotometric investigations. Tissue fragments from paraffin blocks were deparaffinized in xylene, rehydrated and refixed in a formalin solution and incubated in a solution of 0.5 pepsin in 0.25% hydrochloric acid. After filtration through a 70 micron mesh and centrifugation, the cells were smeared upon a glass slide. Comparison between the results obtained with freshly prepared imprints and with pepsin-extracted cells of the same tumor showed that the extraction technique does not influence the Feulgen reaction or the DNA distribution pattern. Investigations carried out on bladder and embryonal carcinomas have demonstrated that the method permits an analysis of histologically or histochemically identified tumor cells within individual tissue areas.     

人早孕胎盘绒毛膜滋养层细胞体外培养模型的建立

目的通过对早孕胎盘绒毛膜滋养层细胞的分离,纯化和培养,寻找一种稳定、简便可获得较高纯度滋养层细胞的培养方法。方法通过胰酶/DNA酶联合消化法对妊娠6-10周绒毛组织进行消化,获得单细胞悬液,比较Per-coll密度梯度离心和淋巴细胞分离液对滋养层细胞的分离纯化效果。含10?S的DMEM/F12培养基培养,并比较是否应用鼠尾胶原对细胞贴壁和生长的影响。通过免疫荧光方法对滋养层细胞进行鉴定。结果经简化Percoll密度梯度离心分离纯化的滋养层细胞纯度高,明显优于淋巴细胞分离液的分离效果(P<0.001);细胞生长表面预先经鼠尾胶原处理后,细胞贴壁良好,分裂生长旺盛。结论利用简化Percoll密度梯度离心法分离细胞,并在应用鼠尾胶原的条件下进行培养,可以获得满意的人绒毛膜滋养层细胞的体外培养模型。     

p53 overexpression in formalin-fixed, paraffin-embedded tissue detected by immunohistochemistry.

Mutation and overexpression of the p53 gene have been noted in a wide range of human cancers and are thought to play a role in malignant transformation. Previously, immunohistochemical detection of p53 has been possible only in fresh-frozen tissues. We examined p53 expression in paraffin-embedded tissues from 50 epithelial ovarian cancers and 25 primary breast cancers with a modified immunohistochemical (IHC) technique developed in this laboratory, using monoclonal antibody (MAb) PAb1801. The 75 cases were selected from a group of patients in whom the expression levels had already been assessed in a fresh-tissue IHC assay. An identical staining reactivity was observed in both formalin-fixed, paraffin-embedded tissue and fresh-frozen tissue in 48 of 50 (96%) epithelial ovarian cancers and in 23 of 25 (92%) primary breast cancers. Immunodetection of p53 in paraffin-embedded tissue blocks will be a useful alternative to the standard fresh-tissue assay and can accurately reflect the level of p53 expression in human tumors.     

Detection of extracellular matrix antigens (fibronectin, laminin, type IV in collagen) in paraffin embedded sections by avidin-biotin-peroxidase complex labelling

An avidin-biotin-peroxidase labelling method was applied to frozen sections and to routine-fixed, paraffin-embedded sections, and compared with immunohistochemical results of stromal antigen detection (type IV collagen, laminin and fibronectin) obtained in parallel on different neoplasias. Lung, breast and gastrointestinal tract tumors were studied. Superimposable sections were obtained from cryostat and formalin-fixed, paraffin-embedded specimens (previously digested by pepsin). The data demonstrate the potential use of immunohistochemical investigation of paraffin-embedded tissues for histological analyses of tumors.     

Evaluation of ethanol-fixed,paraffin-embedded tissues for proteomic applications

We previously reported that ethanol fixation and paraffin embedding of tissues produce excellent histomorphology and good preservation of macromolecules. Here, we present a detailed evaluation of ethanol-fixed tissues for proteomic initiatives. When proteins were extracted from ethanol-fixed, paraffin-embedded prostate tissue, resolved by two-dimensional gel electrophoresis (2-DE), and stained by standard methods, several hundred protein molecules could be detected and successfully analyzed by mass spectrometry. Protein profiles obtained from ethanol-fixed tissues were highly similar to those observed from frozen tissues, in contrast to the poor protein recovery from formalin-fixed material. The protein content of specific cells that were microdissected from ethanol-fixed tissue sections using laser capture microdissection could also be successfully analyzed by 2-DE. We observed that eosin staining of tissue sections had a detrimental effect on protein separation, whereas hematoxylin staining had minimal consequence. In order to illustrate the applicability of ethanol-fixed tissues for proteomic discovery studies, we compared the protein profiles of patient-matched, normal prostatic epithelial cells and invasive adenocarcinoma cells obtained from ethanol-fixed, paraffin-embedded tissues. A number of differentially expressed proteins was discovered and identified by mass spectrometry. Immunohistochemical analyses performed on ethanol-fixed tissue sections were in agreement with the proteomic discovery findings. In light of these results, we conclude that ethanol-fixed tissues can be successfully utilized for proteomic analyses.     

Immunohistochemical detection of tissue factor (TF) on paraffin sections of routinely fixed human tissue

Tissue factor (TF), a 47 kDa transmembrane glycoprotein, is the essential receptor and cofactor for factor VII/VIIa. Its distribution in normal tissues and in tumours has been recently investigated immunohistochemically with monoclonal and polyclonal anti-TF antibodies in frozen sections. The cardinal problem of this technique is the difficulty of determining exactly the localization of the reaction product at least in certain tissues. Here, we demonstrate a method using monoclonal anti-TF antibodies to detect TF in routinely fixed, microwaved, paraffin-embedded tissues. Generally, there were no fundamental differences in TF distribution in frozen and paraffin-embedded material. However, in most cases, the paraffin sections allow a better cellular localization of TF. For example, the staining pattern for TF in both kinds of sections is identical in kidney, brain and skin. The paraffin-embedded material, however, clearly shows that TF expression is restricted to the parietal and the visceral epithelia of Bowman's capsule of glomeruli in the kidney, and to astrocytes and their processes in the brain. TF reactivity in the skin is revealed to be cell membrane-bound; in cardiomyocytes TF shows an exclusively sarcolemmal localization. The immunohistological detection of TF in paraffin sections is a powerful tool for systematic studies on the possible role of TF in the context of physiological and pathological studies.     

Utility of fluorescence in situ hybridization for ploidy and p57 immunostaining in discriminating hydatidiform moles

Discrimination between complete moles (CMs), partial moles (PMs), and hydropic abortions (HAs) is important as the risk of persistent gestational trophoblastic disease (GTD) differs for each condition. We evaluated whether ancillary fluorescence in situ hybridization (FISH) with a set of chromosome enumeration probes (CEP) for chromosomes X, Y, and 17 and p57 immunostaining could improve the clinical diagnosis. Forty-one products of conception (POC) were reclassified according to clinical performance, morphology, p57 immunostaining results, and FISH results. The accuracy of histological examination alone was 85% for the original diagnosis. FISH analysis showed diploidy in 19 of 20 CMs and triploidy in 4 of 6 PMs. The concordance rate was 92.5% on using the CEP probes. p57 Staining was negative in all CMs and positive in all PMs and HAs. Chromosomal abnormality was detected in 3 cases of HA by using FISH. In conclusion, combined p57 immunostaining and FISH with a set of 3 CEP probes for chromosomes X, Y, and 17 could be useful in the classification of hydatidiform moles.     

The use of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections for the study of microchimerism

We describe here a simple and versatile method of fluorescence in situ hybridization (FISH) on paraffin-embedded tissue sections with specific application in the study of microchimerism, that is, the presence of intact foreign cells within an individual. This is accomplished through the use of X and Y chromosome-specific probes to identify the presence of male nuclei within a tissue section from a female, and vice versa. This technique requires only minor modification if at first the hybridization does not yield fluorescent signals of high quality. Analysis of a wide variety of tissue types is possible with this method, and multiple tissue types from one or more individuals can be processed in the same hybridization reaction. This robust FISH method has been used successfully in our laboratory to investigate fetal cell microchimerism in the following paraffin-embedded tissue types: skin, lung, thyroid, adrenal gland, lymph node, heart, spleen, liver, pancreas, kidney, and intestine.