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PubMed1987

Feulgen DNA stainability of bone tumors after demineralization.

Microspectrophotometric DNA analysis of archival bone tumor tissue is often impeded by previous acid demineralization, which destroys Feulgen DNA stainability. To find an alternative to acid for prospective DNA studies of bone tumors in tissue sections, Feulgen stainability of fresh osteosarcoma specimens after demineralization in neutral EDTA was investigated. The reliability of DNA analysis of weakly Feulgen-stained sections from archival tissue was also studied. Demineralization of four fresh specimens in EDTA slightly reduced Feulgen DNA stainability compared to nondemineralized preparations but did not affect the determination of ploidy level. Hydrolysis tests of one diploid and one hyperploid osteosarcoma showed that the staining relationship between control and tumor cells was not altered by EDTA pretreatment. For DNA studies of bone tumors requiring demineralization, EDTA offers a means of retaining nuclear Feulgen stainability. In 22 archival osteosarcoma specimens of varying Feulgen stainability, three different upper limits of light transmission (75, 85, and 95%) were applied to test the significance of background disturbances in relation to nuclear stain intensity. The relationship between the median total extinction of the control and tumor cell populations was not significantly affected by altering the upper transmission limit except in four poorly stained lesions. The control cells of these four specimens exhibited a median total extinction less than one-third of the maximum encountered. The results suggest that weakly stained archival specimens can be tested for selecting those appropriate for ploidy determination.

Bone Neoplasms↗
PubMed1990

Comparison of the Papanicolaou and Feulgen staining methods for DNA quantification by image analysis.

The possibility of using archival cytology material to study the evolution of neoplastic disease with regard to DNA content abnormalities was investigated. The accuracy of measuring the integrity optical density (OD) of nuclei that correlates to DNA amounts of those nuclei, on slides stained by the Papanicolaou method, was assessed and compared with a standard Feulgen method. Our data on rat liver nuclei peritoneal washings from patients with ovarian cystadenofibromas and ovarian cystadenocarcinomas suggested that analysis of cytological material using the Papanicolaou method is not reliable and that destaining the slides followed by Feulgen staining provides an optimal and reliable method of DNA quantification.

Adenofibroma↗
PubMed1990

Variations in Feulgen stainability of epithelial parenchymal cells extracted from paraffin-embedded salivary gland specimens.

The variations of Feulgen stainability of cells extracted from paraffin-embedded archival specimens for DNA assessment by means of image cytometry (ICM) were investigated in normal salivary gland parenchyma. The Feulgen stainability of the deparaffinized, rehydrated, and disaggregated preparations was found to exhibit variations of up to 300%, expressed by the mean of integrated optical density (IOD), when a routine procedure was applied to a first series of Cytospin preparations of disaggregated specimens. When measured in nondisaggregated tissue sections, only negligible variations were observed. After minimization of the mechanical strains to the cellular material in the Cytospin preparations in a second series, the variations in Feulgen stainability were found to be considerably lower. The findings indicate that the main reason for variations in the Feulgen stainability of extracted cells is, most likely, the disaggregation procedure itself. Factors such as initial treatment of the specimens, duration and kind of formalin fixation, and length of storage time periods seem to be of minor importance. Retrospective studies on paraffin-embedded specimens require a carefully controlled tissue type-adapted disaggregation procedure. In addition, we concluded that the interpretation of histograms, obtained by means of ICM DNA assessments in Cytospin preparations of archival material, requires a well-defined internal specific standard.

Coloring Agents↗
PubMed1993

Method for counting mitoses by image processing in Feulgen stained breast cancer sections.

This study describes an image processing method for the assessment of the mitotic count in Feulgen-stained breast cancer sections. The segmentation procedure was optimized to eliminate 95-98% of the nonmitoses, whereas 11% of the mitoses did not survive the segmentation procedure. Contour features and optical density measurements of the remaining objects were computed to allow for classification. Twelve specimens were analyzed, nine used to serve as a training set, and three put aside for later use as independent test set. The fully automatic image processing method correctly classified 81% of the mitoses at the specimen level while inserting 30% false positives. The automatic procedure strongly correlated with the interactive counting procedure (r = 0.98). Although the fully automatic method provided satisfactory results, it is not yet suited for clinical practice. The automated method with an interactive evaluation step gave an accurate reflection of the mitotic count showing an almost perfect correlation with the results of the interactive morphometry (r = 0.998). Therefore this semiautomated method may be useful as prescreening device.

Automation↗
PubMed1994

Practical aspects of fixatives in high resolution nuclear image analysis.

Three different methods of fixation (ethanol/Carbowax, formaldehyde, and Carnoy) and four different protocols (without Böhm post-fixation on an uncoated slide, and with Böhm post-fixation on Poly-L-Lysine coated slide, an uncoated slide and a previously Papanicolaou stained slide) were evaluated for their application in high resolution image analysis of Feulgen stained nuclei. The aim of the study was to find a combination with the best reproducibility and the least variance under normal laboratory conditions. Care was taken not to exclude any "normal" laboratory variability. The combinations were evaluated for densitometric, geometric, as well as texture features. Selected features were determined on a CAS-100 using the Cell Measurement Program (Cell Analysis Systems, Inc. Lombard, IL). Diploid and tetraploid rat liver nuclei were used. Ethanol/Carbowax fixation-with Böhm post-fixation proved most stable. This fixation method also gave feature values for previously Papanicolaou stained slides that were comparable to direct Feulgen stained nuclei. Acceptable results were achieved with all three fixatives and the various combinations if one adhered strictly to protocol. In routine practice this usually does not happen. Therefore Ethanol/Carbowax fixation with Böhm post-fixation was considered most suited for routine determination of feature values on the CAS-100.

Acetates↗
PubMed2003

Aggressive papillary male breast carcinoma on fine-needle cytology sample.

Papillary carcinoma (PC) is a histological variant of breast carcinoma that is more frequently observed in males than in females, showing the same cytological features in both sexes. PC is characterized by a low grade of malignancy and a generally favorable course. We describe a case of male breast PC (MPC) diagnosed by fine-needle cytology (FNC) in which some aggressive morphologically detectable features were associated with bland cytologic features of the tumor. FNC was performed on a 3 cm palpable mass of the left breast of a 55-yr-old male. FNC yielded abundant bloody material. Two smears were Diff-Quik and Papanicolaou stained, others were used for immunocytochemical assessment of estrogen, progesterone, c-erbB-2, and Ki-67; another was Feulgen stained for DNA ploidy. Smears were highly cellular, showing isolated cells and papillary structures. Cells showed tall and well-defined cytoplasm with a columnar aspect, light anisonucleosis, coarse chromatin, and small nucleoli. Immunoperoxidase staining demonstrated positivity for estrogen (50%), negativity for progesterone, intense positivity for c-erbB-2, with specific membrane staining and positivity for Ki-67 in more than 20% of the cells. DNA-ploidy showed an aneuploid histogram with 5c exceeding rate (5cER) of 59% and 2c deviation index (2cDI) of 29%. Subsequent surgical pathology examination confirmed the cytological diagnosis of papillary carcinoma; moreover, it revealed neoplastic endolymphatic thrombi and infiltrative border of the tumor that reached the thoracic wall. Cytological features can suggest diagnosis of MPC on FNC samples. Immunocytochemical evaluation of c-erbB-2 and Ki-67 and DNA ploidy evaluation on cytological smears might reveal a biological aggressiveness of PC despite the bland microscopic features of the tumor and this should influence the therapeutic procedure.

Biomarkers, Tumor↗
PubMed1988

Increased sensitivity for Coomassie staining of sodium dodecyl sulfate-polyacrylamide gels using PhastSystem Development Unit.

Staining of proteins in PhastGel gradient media with Coomassie Blue R 350 was considerably improved using a lower concentration of methanol (10% v/v) and 2% ammonium sulfate in the staining solution and 10% acetic acid for destaining. The detection limit in sodium dodecyl sulfate-polyacrylamide gels was lowered by a factor of 10 to about 2 ng per protein band. The Coomassie staining method was adapted to the newly developed silver staining procedure so that both can be used in parallel in PhastSystem.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

High resolution two-dimensional electrophoresis and sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cheese proteins after rapid solubilisation.

The proteins of cheese are rapidly solubilised by heating to 95 degrees C in buffered 2% sodium dodecyl sulfate, 5% 2-mercaptoethanol. Electrophoretic analysis of the solubilised proteins by either one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis or high resolution two-dimensional electrophoresis yields reproducible patterns characteristic of an individual cheese and its extent of ripening. The patterns reveal (i) the residual amounts of milk casein and whey proteins, and (ii) the appearance of casein degradation products, including pink-violet components as detected by Coomassie Blue staining.

Cheese↗
PubMed1988

Application of Neuhoff's optimized Coomassie brilliant blue G-250/ammonium sulfate/phosphoric acid protein staining to ultrathin polyacrylamide gels on polyester films.

An optimized Coomassie staining procedure, utilizing Coomassie Brilliant Blue G-250 in phosphoric acid/ammonium sulfate, was applied to ultrathin-layer isoelectric focusing in 0.18 mm polyacrylamide gels, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in 0.38 mm polyacrylamide gels, both backed to Gel-Fix polyester supporting films. After isoelectric focusing staining of gelatin and acidic proteins was better with the phosphoric acid/ammonium sulfate procedure than with conventional organic solvent methods. When applied to gels after sodium dodecyl sulfate-polyacrylamide gel electrophoresis the sensitivity of the phosphoric acid/ammonium sulfate method was equal to that on conventional staining but lower than on silver staining.

Acrylic Resins↗
PubMed1988

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.

Acrylic Resins↗
PubMed1990

Suitability of staining techniques for the detection and quantitation of nonhistone high mobility group proteins.

Three different staining techniques were compared for the detection of nonhistone high mobility group (HMG) proteins after acidic urea-polyacrylamide gel electrophoresis. Silver staining after glutaraldehyde fixation provides the highest detection sensitivity. Because of the acid solubility of HMG proteins special care has to be taken concerning fixation. Staining with colloidal CBB G-250 according to Neuhoff et al. is superior in sensitivity and reliability of quantitation when compared with noncolloidal Coomassie Brilliant Blue R-250. High detection sensitivity and reproducibility of quantitation are prerequisites for studying the tissue-specific expression of HMG proteins. In the present study tissue-specific differences in the molar amounts of various HMG proteins in thymus and erythrocytes of the chicken are documented by application of the methods tested.

Animals↗
PubMed1990

Alternative methods for fixing and staining gliadins in polyacrylamide gels.

Staining efficiencies of Coomassie Brilliant Blue G-250 (CBB G-250) and Coomassie Brilliant Blue R-250 (CBB R-250) in various media were studied in efforts to reduce or eliminate requirements for trichloroacetic acid (TCA). Stained gels were compared with gels stained with CBB R-250 in 12% TCA and evaluated for overall stain and background. Because of qualitative effects, stain intensities of low- and high-mobility gliadins were also evaluated. Results indicated gliadins are fixed under a wide range of conditions, permitting adjustment of conditions to provide optimum staining. CBB G-250 and R-250 in tap water fixed and stained most gliadins. Best results were obtained with CBB G-250 in 2% TCA, in 2% TCA containing 5% sodium sulfate, and in 2% and 5% phosphoric acid containing 5% sodium chloride or 5% sodium sulfate. Gels stained in these media were more easily observed during staining and more easily destained than gels stained in CBB R-250 in 12% TCA.

Electrolytes↗
PubMed1990

Isoelectric focusing of human parotid salivary proteins in hybrid carrier ampholyte-immobilized pH gradient polyacrylamide gels.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.

Acrylic Resins↗
PubMed1990

Two-dimensional gel electrophoresis, protein electroblotting and microsequencing: a direct link between proteins and genes.

We have used two-dimensional gel electrophoresis as a general "preparative" method to purify proteins for microsequencing analysis. In the first experiments, proteins derived from a total extract of Nicotiana tabacum leaf tissue were directly blotted from the gel onto poly(4-vinyl-N-methylpyridinium iodide)-coated glass fiber sheets. The major spots were excised and subjected to NH2-terminal sequence analysis, which made it possible to identify five of the eight selected proteins, while two more were recognized by generated internal sequences. In a second set of experiments, proteins of human origin were separated on multiple two-dimensional gels and the Coomassie Brilliant Blue-stained spots were excised from the gels. The combined spots were re-eluted and concentrated in a new gel and blotted on Immobilon. They were fragmented by in situ proteolysis and the generated peptides were separated by reverse phase-high performance liquid chromatography and sequenced. At the average, the internal sequences that were obtained covered 35 residues per protein and allowed unambiguous identification of 13 of the 23 proteins analyzed so far. The sequence information obtained of the unidentified proteins is sufficient for further cloning. These results demonstrate that systematic sequence analysis of the major proteins seen in two-dimensional gels is within the reach of current technologies. This offers a unique opportunity to link information contained in protein databases with known or forthcoming DNA sequence data.

Amino Acid Sequence↗
PubMed1990

An improved scheme for the identification of antigens recognized by specific antibodies in two-dimensional gel electrophoresis and immunoblotting.

This paper describes an improved scheme for the identification of antigens in crude extracts recognized by specific antibodies when analyzed by a combination of two-dimensional gel electrophoresis and immunoblotting. First, protein components in gels are electrophoretically transferred to a polyvinylidene difluoride membrane which does not shrink or change dimensions in organic solvents. The efficiency of transfer and the localization of sample proteins on the membrane are checked and recorded by staining the blotting membrane with Fast Green FCF and recording the profile on a transparency. After blocking and the immunoassay, the results are recorded by photography. The sites of immune reaction are marked and the same membrane is restained briefly with Coomassie Brilliant Blue R-250 for the protein profile. Thus antigens in complex mixtures, recognized by antibodies of interest, can easily be identified from the restained membrane. If the whole protein profile is not well demonstrated, when used in combination with the profile recorded on the transparency, spots appearing on the restained membrane can still be used as useful landmarks in the final unequivocal antigenic identification. This improved scheme circumvents problems arising from membrane shrinkage and difficulties in accurately matching immunoreactive spots by conventional procedures and thus provides an accurate, simple and fast approach in the identification of antigens after immunoblotting.

Antibodies, Monoclonal↗
PubMed1990

Development of a database of amino acid sequences for human colon carcinoma proteins separated by two-dimensional polyacrylamide gel electrophoresis.

The tandem use of preparative two-dimensional polyacrylamide gel electrophoresis (2-DE) and electroblotting onto polyvinylidene difluoride membranes has been employed to rapidly isolate a number of proteins from a crude cell extract of a human colon carcinoma cell line (LIM 1863). The immobilized proteins were located by staining with Coomassie Brilliant Blue R-250, and selected protein spots were excised and subjected to Edman degradation. Our results demonstrate that overall sequence yields in the 3-20 pmol range can be achieved on protein spots from four identical 2-DE gels; approximately 150-200 micrograms of total protein was applied to a single 2-DE gel. An approximate two-fold increase in sensitivity of phenylthiohydantoin-amino acid detection (subpicomole range) was achieved by fitting our commercial sequencers with a simple sample transfer device which permitted the analysis of the total phenylthiohydantoin-amino acid derivative. N-Terminal amino acid sequence data was obtained for thirteen electroblotted proteins. All of these sequences positively matched those of proteins of known structure listed in the available protein sequence databases. Approximately 40% of the electroblotted proteins did not yield N-terminal sequence information, presumably because they had blocked N-termini (either naturally or artifactually). Internal amino acid sequence information was obtained from three proteins isolated by preparative 2-DE. This was achieved by in situ digestion of the proteins in the gel matrix with Staphylococcus aureus V8 protease, electrophoresis of the generated peptides in a one-dimensional gel, electrotransfer of the peptides to a polyvinylidene difluoride membrane and microsequence analysis of the electroblotted peptides.

Amino Acid Sequence↗