Chapter 9 Image Analysis of Chromatin Remodelling Benedicto de Campos Vidal, Marina B. Felisbino, and Maria Luiza S. Mello Abstract Chromatin packaging plays a significant role in regulating gene transcription. Study of the higher-order packing states of chromatin by image analysis at the light microscope level, especially when validated by methods of molecular biology, immunochemistry, and/or immunocytochemistry, enabled the detection of changes involved in the processes associated with or preceding alterations in transcriptional activities. Here, we recommend and describe the use of relatively simple methods for staining and detecting chromatin remodelling by image analysis. Key words Chromatin structure, Chromatin supraorganization, Epigenetics, Feulgen staining, Image analysis, DNA, Histones, Histone deacetylases
1
Introduction Chromatin is a complex of DNA, histones, nonhistone proteins, and RNA that is located in the nucleus of a cell in interphase. The packaging helps to regulate gene transcription. Increasing hierarchical levels of chromatin packing states results in the chromatin supraorganization that can be viewed using a light microscope and is currently studied with image analysis in association with biochemical and topochemical assays. The patterns of distribution and the organization of different higher-order chromatin packing states may characterize particular nuclear phenotypes and their changes under normal, physiological situations as well as experimental conditions [1–4]. Some examples of chromatin remodelling that are revealed with image analysis in preparations where DNA is topochemically stained are the following: 1. Chromatin decondensation in HeLa and NIH 3T3 cells following exposure of the cells to specific histone deacetylase class I inhibitors such as valproic acid and trichostatin A [5, 6] (Fig. 1);
Juan C. Stockert et al. (eds.), Functional Analysis of DNA and Chromatin, Methods in Molecular Biology, vol. 1094, DOI 10.1007/978-1-62703-706-8_9, © Springer Science+Business Media New York 2014
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Fig. 1 False-colored images of Feulgen-stained NIH 3T3 cells. The condensed chromatin areas, shown as green points in the untreated control (a), become less abundant in cells treated with valproic acid, which inhibits histone deacetylases and promotes the acetylation of histones H3 and H4 (b). Red points correspond to gray values lower than the value established for segmentation of the areas with condensed chromatin, but higher than the value for the nuclear background
this finding was accompanied by the induction of epigenetic changes associated with hyperacetylation of histones H3 and H4, which was demonstrated biochemically [5–9]. 2. Increasing homogeneity of chromatin packaging with increasing polyploidy in hepatocytes from hyperglycemic non-obese diabetic mice [10] and more heterogeneously distributed condensed chromatin with increasing polyploidy in hepatocytes from normoglycemic aged mice [10, 11]; these results have been validated using micrococcal nuclease digestion assays that assess chromatin accessibility [10]. 3. Chromatin condensation associated with changes in epigenetic marks and posttranscriptional modifications of RNA in the breast epithelium of postmenopausal women, which explain the mechanisms that mediate the protective effects associated with early full-term pregnancy and the development of breast cancer at menopause [12]. 4. Chromatin condensation associated with apoptosis in bullfrog erythrocytes and benzo[a]pyrene-transformed human breast epithelial cells [13]. 5. Increasing chromatin condensation accompanying the transformation of NIH 3T3 cells by the c-Ha-ras oncogene [1, 14] and the expression of different stages of the in vitro tumorigenesis process in benzo[a]pyrene- or 17-β-estradioltransformed human breast epithelial cells [2, 15–17].
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Fig. 2 A representative scatter diagram demonstrating the distribution of the different nuclear phenotypes defined in terms of condensed chromatin area relative to the nuclear area (Sc %) versus the contrast between condensed and total chromatin (AAR), as modified from Vidal [20]
6. An increasing degree of condensation in more loosened packed chromatin areas in hepatocytes under starvation conditions that are associated with a decrease in the formation of extended chromatin fibers under the action of gravity [18]. Chromatin remodelling can be assessed by matching specific parameters obtained by image analysis procedures of Feulgenstained cells using an automatic scanning microspectrophotometry or video system (Figs. 2 and 3). Alternatively, changes in chromatin packing states have been equally revealed by evaluating specific, isolated parameters by video image analysis, such as absorbance variability per nucleus image, entropy, energy, and contrast, preferentially in Feulgen-stained preparations but also in hematoxylinstained nuclei [12]. DNA in any state of chromatin packaging can be visualized in cell preparations using the Feulgen reaction [19]. Heterogeneous chromatin packing states are maintained during a rapid acetic ethanol fixation and the Feulgen acid hydrolysis, provided that the hydrolysis times do not exceed the periods which are required for maximal DNA depurination [19]. The chromatin remodelling approaches highlighted here have been validated with current molecular biology, immunochemical or immunocytochemical assays.
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Fig. 3 A scatter diagram of Sc % versus AAR for Feulgen-stained NIH 3T3 cells. The nuclei from the cells treated with 1.0 mM valproic acid for 2 h are shown in red and the untreated controls are shown in black. This representation indicates that during treatment with a histone deacetylase inhibitor like the valproic acid a decrease in the area covered by condensed chromatin is observed along with a simultaneous increase in chromatin decondensation (n, 80) [5, 6]
2
Materials All of the reagents are of analytical grade.
2.1 Glass and Other Materials
1. Bunsen burner. 2. Heat-resistant glass Erlenmeyer flask. 3. Glass cylinders. 4. Glass filter. 5. Whatman filter paper. 6. Glass or plastic slides and coverslips. 7. Coplin jars with covers or Petri dishes with covers. 8. Borels. 9. Stoppered bottle. 10. Pipettes.
2.2
Solutions
1. Freshly prepared fixative: absolute ethanol-glacial acetic acid (3:1, v/v) (see Note 1). 2. 70 % ethanol. 3. Concentrated HCl solution (see Note 2). 4. 1 M HCl.
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5. 0.1 M HCl at 4 °C. 6. Schiff’s reagent preparation: (a) Boil 100 mL distilled water in an Erlenmeyer flask using a Bunsen burner. (b) Stop heating and add 1 g of basic fuchsin (or pararosaniline) to the water. (c) Cool solution to 60 °C and filter. (d) Add 4 g of sodium bisulfite and dissolve well. (e) Add 10 mL of 1 M HCl. (f) Let the solution stand in the dark for 24 h in a stoppered bottle. (g) Add 200 mg of activated charcoal. (h) Shake the solution strongly and then filter two to three times to remove the charcoal. (i) Store at 4 °C. The solution must be colorless or a pale yellow straw color. If the solution acquires a pink color, it should be discarded. 7. Freshly prepared sulfurous water (10 % sodium metabisulfite: 1 M HCl: distilled water, 1:1:18, v/v/v). 8. Xylene. 9. Natural Canada balsam or Cargille oil (R.P. Cargille Lab., Cedar Grove, USA) (nD = 1.54). 2.3 Cellular Materials 2.4
Equipment
2.4.1 Automatic Scanning Microspectrophotometry
2.4.2 Video Image Analyzer
1. Prepare imprints, smears, or isolated cells according to the study purposes (see Note 3). An automatic scanning microspectrophotometer (e.g., Carl Zeiss, Oberkochen, Germany—see Note 4) interfaced to a personal computer and respective software that controls the predominantly unidirectional scanning motion of the stage and informs the user of the image analysis parameters. The equipment requirements include the following: a halogen 100-W/12-V lamp, a stabilized electronic power supply, a light modulator, an adequate photomultiplier, λ = 565 nm obtained with a monochromator filter ruler or an interference filter, and a scanning spot size of 0.5 × 0.5 μm. Other requirements, which vary as a function of the size of the nuclei to be measured, include the following: Planapo objectives, optovar, measuring diaphragm diameter, field diaphragm diameter, and LD-Epiplan condenser magnifications (see Note 5). A photomicroscope (e.g., Carl Zeiss Axiophot 2, Olympus BX51-B) that feeds the images to a computer through a color video camera, equipped with the image analysis software (e.g., Kontron KS400-3, Munich, Germany; Image ProPlus 6.3, Media
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Cybernetics, Inc., Bethesda, USA). The equipment requirements also include a halogen 100-W/12-V lamp, a voltage regulator maintained at a constant point selected by the operator, a luminous-filter diaphragm at its maximal opening, filter wheels rotated into the open position, a 0.90 condenser, and a λ value of 565 nm interference filter (see Note 6). Other conditions, which vary as a function of the size of the nuclei to be measured, include Neofluar or UPlanFl objectives and optovar magnifications (see Note 7).
3
Methods
3.1 Staining Procedure
1. Immediately after preparing the samples, fix them in absolute ethanol-glacial acetic acid for 1 min at room temperature (see Note 1). Then rinse the preparations in 70 % ethanol for 5 min and air dry. 2. Next, subject the samples to the Feulgen reaction [19]. First, subject the preparations to an acid hydrolysis in HCl. Acid concentration and temperature depend on the researcher’s preference. The hydrolysis time should be established after a preliminary test (see Note 2). Then, stop the hydrolysis treatment by subjecting the preparations to a rapid wash in cold 0.1 M HCl (4 °C) followed by a 40-min treatment with Schiff’s reagent at room temperature. Transfer Schiff’s reagent from the stoppered bottle to the Coplin jar or borel using a pipette, taking care to avoid touching the bottom or the sides of the bottle to prevent contamination of the solution. 3. Transfer the preparations into three washes of sulfurous water for 5 min each, and then, rinse the slides in distilled water and air dry. Clear the preparations in xylene, and mount them in natural Canada balsam (nD = 1.54) (see Note 8). The DNA will stain a magenta color. Allow the mounting medium to completely dry. If using cells cultivated on plastic slides, mount dry-stained preparations in Cargille oil (R.P. Cargille Lab., Cedar Grove, USA) with nD = 1.54. 4. Keep the slides in the dark to avoid fading of the staining reaction.
3.1.1 Automatic Scanning Microspectrophotometry
1. To obtain Feulgen-stained nuclear images, select the equipment conditions that are appropriate for the size of the nuclei to be measured (see Note 5) and calibrate the light transmittance of the background to 100 %. 2. Consider individual measuring points showing a very low absorbance (e.g., 0.020) as background, and set the computer system to automatically remove these points from the nuclear image.
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3. A preliminary test to select an absorbance value threshold (cutoff point) should be performed to identify the areas covered by condensed chromatin. This value should remain constant during all of the experiments (see Note 9). 4. Image analysis parameters According to Vidal [20], to construct a scatter diagram where each point represents a nucleus, it is necessary to determine the parameters that demonstrate a contrast between condensed chromatin and total chromatin (AAR) [21] and the area covered by condensed chromatin relative to the nuclear area (Sc %). These parameters are obtained following the calculations of other parameters as follows: 1. AT, total integrated absorbance (in this case, nuclear Feulgen-DNA values in arbitrary units). 2. Ac, integrated absorbance over the preselected cutoff point, which corresponds to the Feulgen-DNA values of the condensed chromatin. 3. Ac %, condensed chromatin Feulgen-DNA values relative to whole-chromatin Feulgen-DNA values 4. ST, nuclear absorption area in μm2. 5. Sc, area in μm2 covered with stained chromatin displaying the absorbances above the selected cutoff point mentioned in item 3. 6. Sc %, area covered by condensed chromatin relative to the nuclear area. 7. AAR = (Ac/Sc)/(AT/ST), average absorption ratio, a dimensionless parameter that reports how many times the average absorbance of the condensed chromatin exceeds that of the entire nucleus [21]. 3.1.2 Video Image Analysis
The software that accompanies each type of image analyzer provides researchers with the process of image acquisition, segmentation, and featuring [22]. Black-and-white images are digitized so that a value on a scale of 256 shades of gray could be assigned. All image pixels with a gray level are given values between 0 (black) and 255 (white). The pixels are the lowest hierarchic level objects; they carry the value and coordinates of the features. Different gray levels are converted to false-color (= pseudocolor) images to facilitate area integration [22] (Fig. 1). Generally there is a list of geometric, densitometric, and textural parameters that could be selected by the researcher on the basis of his/her needs. Some image analyzers also allow researchers to construct parameters of their choice [22]. The most common features are nuclear area, nuclear absorbing area, nuclear perimeter, roundness, mean gray value per nucleus (which may be converted into absorbance (optical density, OD)), absorbance variability per nucleus, contrast, entropy, energy, minimum feret, maximal feret, feret ratio, and many others.
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A feret diameter is the distance between two parallel lines drawn tangentially to opposite sides of an object boundary [23]. The maximal feret corresponds to the largest long axis of the object; the minimal feret corresponds to the shortest width axis perpendicular to the largest long axis of the object [23]. The feret ratio (minimal feret/maximal feret) is an indicator of the elongation of the absorbing object. The integrated OD (= IOD) is equal to the absorbance multiplied by the nuclear absorbing area. For Feulgen-stained nuclei, IOD represents Feulgen-DNA values in arbitrary units (see Notes 10 and 11).
4
Notes 1. An absolute ethanol-glacial acetic acid mixture is the best fixative for DNA studies that utilize the Feulgen reaction. 2. In our laboratory, we ordinarily use 4 M HCl at room temperature for ethanol-acetic acid-fixed materials. Because the hydrolysis time varies according to the acid concentration and temperature as well as the material specificities, time course should be performed to determine the time that is mostly adequate for maximal DNA depurination, that is, that which provides the largest staining intensity. 3. Paraffin sections may be used for establishing Feulgen-DNA values provided the sections are thick enough to contain whole nuclei for measurements. If hematoxylin-stained sections are used to study the degree of compactness and the distribution of chromatin condensed areas, special care should be taken and a larger sample size should be used. 4. It is recommended that the microspectrophotometer construction characteristics reduce glare effects and diffraction errors. 5. We use Planapo objectives 63/0.90 or 40/0.95, optovar 2.0, measuring diaphragm 0.25 mm, and LD-Epiplan 16/0.32 condenser to analyze nuclei from mammalian hepatocytes, cardiomyocytes, mammalian culture cells, and nucleated erythrocytes. An R-928 photomultiplier is a component of a Zeiss automatic scanning microspectrophotometer. 6. In our experience, an interference filter of λ = 546 nm is also adequate for measuring the Feulgen-stained response if the interference filter of λ = 565 nm is not available. 7. We use Neofluar or UPlanF1 objectives 40/0.75 and optovar 2.0 for nuclei from mammalian hepatocytes, mammalian culture cells, nucleated erythrocytes, and insect cells.
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8. The Feulgen reaction should not be followed by any treatment with ethanol solutions; otherwise, the staining will become partially removed. 9. Sometimes, more than one cutoff point can be selected (e.g., 0.100, 0.200) to obtain results with a better capacity for envisioning different chromatin condensation degrees [1, 14, 22]. 10. Arbitrary units can be converted into absolute units (e.g., DNA picograms, 10−12 g) if a control with a known DNA concentration is used. 11. To study the changes in chromatin packing states, the best features for comparisons are absorbance variability per nucleus, contrast, entropy, and energy.
Acknowledgments This work was supported by the São Paulo State Research Foundation (FAPESP, grants no. 2010/50015-6 and 2009/11763-0) and the Brazilian National Council on Development and Research (CNPq). References 1. Mello MLS, Contente S, Vidal BC et al (1995) Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis. Exp Cell Res 220:374–382 2. Vidal BC, Russo J, Mello MLS (1998) DNA content and chromatin texture of benzo[a] pyrene-transformed human breast epithelial cells as assessed by image analysis. Exp Cell Res 244:77–82 3. Gilbert N, Gilchrist S, Bickmore WA (2005) Chromatin organization in the mammalian nucleus. Int Rev Cytol 242:283–336 4. Cremer T, Zakhartchenko V (2011) Nuclear architecture in developmental biology and cell specialization. Reprod Fertil Dev 23:94–114 5. Felisbino MB, Tamashiro WMSC, Mello MLS (2011) Chromatin remodeling, cell proliferation and cell death in valproic acid-treated HeLa cells. PLoS One 6:e29144 6. Felisbino MB (2012) Remodelação cromatínica, anomalias cromossômicas e morte celular em condições de inibição de deacetilases de histonas em células HeLa e NIH 3T3. Masters’ dissertation, University of Campinas, Campinas, p 106 7. Tóth KF, Knoch TA, Wachsmuth M et al (2004) Trichostatin A-induced histone acetylation
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causes decondensation of interphase chromatin. J Cell Sci 117:4277–4287 Rao J, Bliattacharya D, Banerjee B et al (2007) Trichostatin-A induces differential changes in histone protein dynamics and expression in HeLa cells. Biochem Biophys Res Commun 363:263–268 Sami S, Höti N, Xu HM et al (2008) Valproic acid inhibits the growth of cervical cancer both in vitro and in vivo. J Biochem 144:357–362 Ghiraldini FG, Silva IS, Mello MLS (2012) Polyploidy and chromatin remodeling in hepatocytes from insulin-dependent diabetic and normoglycemic aged mice. Cytometry A 81A: 755–764 Moraes AS, Guaraldo AMA, Mello MLS (2007) Chromatin supraorganization and extensibility in mouse hepatocytes with development and aging. Cytometry A 71:28–37 Russo J, Santucci-Pereira J, de Cicco RL et al (2012) Pregnancy-induced chromatin remodeling in the breast of postmenopausal women. Int J Cancer 131:1059–1070 Mello MLS (2007) Discrimination of apoptotic cells by image analysis. In: Corvin AJ (ed) New developments in cell apoptosis research. Nova Sci. Publ., Inc., New York, pp 273–287
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14. Mello MLS, Russo J (1990) Image analysis of Feulgen-stained c-H-ras-transformed NIH/3T3 cells. Biochem Cell Biol 68: 1026–1031 15. Mello MLS, Russo P, Russo JLL (2007) 17-β-estradiol affects nuclear image properties in MCF-10F human breast epithelial cells with tumorigenesis. Oncol Rep 18:1475–1481 16. Mello MLS, Vidal BC, Russo IH et al (2007) DNA content and chromatin texture of human breast epithelial cells transformed with 17-β-estradiol and treated with the estrogen antagonist ICI182,780 as assessed by image analysis. Mutat Res 617:1–7 17. Mello MLS, Russo P, Russo JLL (2009) Entropy of Feulgen-stained 17-β-estradioltransformed human breast epithelial cells as assessed by restriction enzymes and image analysis. Oncol Rep 21:1483–1487 18. Moraes AS, Vidal BC, Guaraldo AMA et al (2005) Chromatin supraorganization and
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extensibility in mouse hepatocytes following starvation and refeeding. Cytometry A 63: 94–107 Mello MLS (1997) Cytochemistry of DNA, RNA and nuclear proteins. Braz J Genet 20:257–264 Vidal BC (1984) Polyploidy and nuclear phenotypes in salivary glands of the rat. Biol Cell 50:137–146 Vidal BC, Schlüter G, Moore GW (1973) Cell nucleus pattern recognition: influence of staining. Acta Cytol 17:510–521 Mello MLS, Vidal BC, Planding W et al (1994) Image analysis: video system adequacy for the assortment of nuclear phenotypes based on chromatin texture evaluation. Acta Histochem Cytochem 27:23–31 Marchevsky AM, Erler BS (1994) Morphometry in Pathology. In: Marchevsky AM, Bartels PH (eds) Image analysis: a primer for pathologists. Raven Press Ltd, New York
1
Introduction Chromatin is a complex of DNA, histones, nonhistone proteins, and RNA that is located in the nucleus of a cell in interphase. The packaging helps to regulate gene transcription. Increasing hierarchical levels of chromatin packing states results in the chromatin supraorganization that can be viewed using a light microscope and is currently studied with image analysis in association with biochemical and topochemical assays. The patterns of distribution and the organization of different higher-order chromatin packing states may characterize particular nuclear phenotypes and their changes under normal, physiological situations as well as experimental conditions [1–4]. Some examples of chromatin remodelling that are revealed with image analysis in preparations where DNA is topochemically stained are the following: 1. Chromatin decondensation in HeLa and NIH 3T3 cells following exposure of the cells to specific histone deacetylase class I inhibitors such as valproic acid and trichostatin A [5, 6] (Fig. 1);
Juan C. Stockert et al. (eds.), Functional Analysis of DNA and Chromatin, Methods in Molecular Biology, vol. 1094, DOI 10.1007/978-1-62703-706-8_9, © Springer Science+Business Media New York 2014
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Fig. 1 False-colored images of Feulgen-stained NIH 3T3 cells. The condensed chromatin areas, shown as green points in the untreated control (a), become less abundant in cells treated with valproic acid, which inhibits histone deacetylases and promotes the acetylation of histones H3 and H4 (b). Red points correspond to gray values lower than the value established for segmentation of the areas with condensed chromatin, but higher than the value for the nuclear background
this finding was accompanied by the induction of epigenetic changes associated with hyperacetylation of histones H3 and H4, which was demonstrated biochemically [5–9]. 2. Increasing homogeneity of chromatin packaging with increasing polyploidy in hepatocytes from hyperglycemic non-obese diabetic mice [10] and more heterogeneously distributed condensed chromatin with increasing polyploidy in hepatocytes from normoglycemic aged mice [10, 11]; these results have been validated using micrococcal nuclease digestion assays that assess chromatin accessibility [10]. 3. Chromatin condensation associated with changes in epigenetic marks and posttranscriptional modifications of RNA in the breast epithelium of postmenopausal women, which explain the mechanisms that mediate the protective effects associated with early full-term pregnancy and the development of breast cancer at menopause [12]. 4. Chromatin condensation associated with apoptosis in bullfrog erythrocytes and benzo[a]pyrene-transformed human breast epithelial cells [13]. 5. Increasing chromatin condensation accompanying the transformation of NIH 3T3 cells by the c-Ha-ras oncogene [1, 14] and the expression of different stages of the in vitro tumorigenesis process in benzo[a]pyrene- or 17-β-estradioltransformed human breast epithelial cells [2, 15–17].
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Fig. 2 A representative scatter diagram demonstrating the distribution of the different nuclear phenotypes defined in terms of condensed chromatin area relative to the nuclear area (Sc %) versus the contrast between condensed and total chromatin (AAR), as modified from Vidal [20]
6. An increasing degree of condensation in more loosened packed chromatin areas in hepatocytes under starvation conditions that are associated with a decrease in the formation of extended chromatin fibers under the action of gravity [18]. Chromatin remodelling can be assessed by matching specific parameters obtained by image analysis procedures of Feulgenstained cells using an automatic scanning microspectrophotometry or video system (Figs. 2 and 3). Alternatively, changes in chromatin packing states have been equally revealed by evaluating specific, isolated parameters by video image analysis, such as absorbance variability per nucleus image, entropy, energy, and contrast, preferentially in Feulgen-stained preparations but also in hematoxylinstained nuclei [12]. DNA in any state of chromatin packaging can be visualized in cell preparations using the Feulgen reaction [19]. Heterogeneous chromatin packing states are maintained during a rapid acetic ethanol fixation and the Feulgen acid hydrolysis, provided that the hydrolysis times do not exceed the periods which are required for maximal DNA depurination [19]. The chromatin remodelling approaches highlighted here have been validated with current molecular biology, immunochemical or immunocytochemical assays.
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Fig. 3 A scatter diagram of Sc % versus AAR for Feulgen-stained NIH 3T3 cells. The nuclei from the cells treated with 1.0 mM valproic acid for 2 h are shown in red and the untreated controls are shown in black. This representation indicates that during treatment with a histone deacetylase inhibitor like the valproic acid a decrease in the area covered by condensed chromatin is observed along with a simultaneous increase in chromatin decondensation (n, 80) [5, 6]
2
Materials All of the reagents are of analytical grade.
2.1 Glass and Other Materials
1. Bunsen burner. 2. Heat-resistant glass Erlenmeyer flask. 3. Glass cylinders. 4. Glass filter. 5. Whatman filter paper. 6. Glass or plastic slides and coverslips. 7. Coplin jars with covers or Petri dishes with covers. 8. Borels. 9. Stoppered bottle. 10. Pipettes.
2.2
Solutions
1. Freshly prepared fixative: absolute ethanol-glacial acetic acid (3:1, v/v) (see Note 1). 2. 70 % ethanol. 3. Concentrated HCl solution (see Note 2). 4. 1 M HCl.
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5. 0.1 M HCl at 4 °C. 6. Schiff’s reagent preparation: (a) Boil 100 mL distilled water in an Erlenmeyer flask using a Bunsen burner. (b) Stop heating and add 1 g of basic fuchsin (or pararosaniline) to the water. (c) Cool solution to 60 °C and filter. (d) Add 4 g of sodium bisulfite and dissolve well. (e) Add 10 mL of 1 M HCl. (f) Let the solution stand in the dark for 24 h in a stoppered bottle. (g) Add 200 mg of activated charcoal. (h) Shake the solution strongly and then filter two to three times to remove the charcoal. (i) Store at 4 °C. The solution must be colorless or a pale yellow straw color. If the solution acquires a pink color, it should be discarded. 7. Freshly prepared sulfurous water (10 % sodium metabisulfite: 1 M HCl: distilled water, 1:1:18, v/v/v). 8. Xylene. 9. Natural Canada balsam or Cargille oil (R.P. Cargille Lab., Cedar Grove, USA) (nD = 1.54). 2.3 Cellular Materials 2.4
Equipment
2.4.1 Automatic Scanning Microspectrophotometry
2.4.2 Video Image Analyzer
1. Prepare imprints, smears, or isolated cells according to the study purposes (see Note 3). An automatic scanning microspectrophotometer (e.g., Carl Zeiss, Oberkochen, Germany—see Note 4) interfaced to a personal computer and respective software that controls the predominantly unidirectional scanning motion of the stage and informs the user of the image analysis parameters. The equipment requirements include the following: a halogen 100-W/12-V lamp, a stabilized electronic power supply, a light modulator, an adequate photomultiplier, λ = 565 nm obtained with a monochromator filter ruler or an interference filter, and a scanning spot size of 0.5 × 0.5 μm. Other requirements, which vary as a function of the size of the nuclei to be measured, include the following: Planapo objectives, optovar, measuring diaphragm diameter, field diaphragm diameter, and LD-Epiplan condenser magnifications (see Note 5). A photomicroscope (e.g., Carl Zeiss Axiophot 2, Olympus BX51-B) that feeds the images to a computer through a color video camera, equipped with the image analysis software (e.g., Kontron KS400-3, Munich, Germany; Image ProPlus 6.3, Media
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Cybernetics, Inc., Bethesda, USA). The equipment requirements also include a halogen 100-W/12-V lamp, a voltage regulator maintained at a constant point selected by the operator, a luminous-filter diaphragm at its maximal opening, filter wheels rotated into the open position, a 0.90 condenser, and a λ value of 565 nm interference filter (see Note 6). Other conditions, which vary as a function of the size of the nuclei to be measured, include Neofluar or UPlanFl objectives and optovar magnifications (see Note 7).
3
Methods
3.1 Staining Procedure
1. Immediately after preparing the samples, fix them in absolute ethanol-glacial acetic acid for 1 min at room temperature (see Note 1). Then rinse the preparations in 70 % ethanol for 5 min and air dry. 2. Next, subject the samples to the Feulgen reaction [19]. First, subject the preparations to an acid hydrolysis in HCl. Acid concentration and temperature depend on the researcher’s preference. The hydrolysis time should be established after a preliminary test (see Note 2). Then, stop the hydrolysis treatment by subjecting the preparations to a rapid wash in cold 0.1 M HCl (4 °C) followed by a 40-min treatment with Schiff’s reagent at room temperature. Transfer Schiff’s reagent from the stoppered bottle to the Coplin jar or borel using a pipette, taking care to avoid touching the bottom or the sides of the bottle to prevent contamination of the solution. 3. Transfer the preparations into three washes of sulfurous water for 5 min each, and then, rinse the slides in distilled water and air dry. Clear the preparations in xylene, and mount them in natural Canada balsam (nD = 1.54) (see Note 8). The DNA will stain a magenta color. Allow the mounting medium to completely dry. If using cells cultivated on plastic slides, mount dry-stained preparations in Cargille oil (R.P. Cargille Lab., Cedar Grove, USA) with nD = 1.54. 4. Keep the slides in the dark to avoid fading of the staining reaction.
3.1.1 Automatic Scanning Microspectrophotometry
1. To obtain Feulgen-stained nuclear images, select the equipment conditions that are appropriate for the size of the nuclei to be measured (see Note 5) and calibrate the light transmittance of the background to 100 %. 2. Consider individual measuring points showing a very low absorbance (e.g., 0.020) as background, and set the computer system to automatically remove these points from the nuclear image.
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3. A preliminary test to select an absorbance value threshold (cutoff point) should be performed to identify the areas covered by condensed chromatin. This value should remain constant during all of the experiments (see Note 9). 4. Image analysis parameters According to Vidal [20], to construct a scatter diagram where each point represents a nucleus, it is necessary to determine the parameters that demonstrate a contrast between condensed chromatin and total chromatin (AAR) [21] and the area covered by condensed chromatin relative to the nuclear area (Sc %). These parameters are obtained following the calculations of other parameters as follows: 1. AT, total integrated absorbance (in this case, nuclear Feulgen-DNA values in arbitrary units). 2. Ac, integrated absorbance over the preselected cutoff point, which corresponds to the Feulgen-DNA values of the condensed chromatin. 3. Ac %, condensed chromatin Feulgen-DNA values relative to whole-chromatin Feulgen-DNA values 4. ST, nuclear absorption area in μm2. 5. Sc, area in μm2 covered with stained chromatin displaying the absorbances above the selected cutoff point mentioned in item 3. 6. Sc %, area covered by condensed chromatin relative to the nuclear area. 7. AAR = (Ac/Sc)/(AT/ST), average absorption ratio, a dimensionless parameter that reports how many times the average absorbance of the condensed chromatin exceeds that of the entire nucleus [21]. 3.1.2 Video Image Analysis
The software that accompanies each type of image analyzer provides researchers with the process of image acquisition, segmentation, and featuring [22]. Black-and-white images are digitized so that a value on a scale of 256 shades of gray could be assigned. All image pixels with a gray level are given values between 0 (black) and 255 (white). The pixels are the lowest hierarchic level objects; they carry the value and coordinates of the features. Different gray levels are converted to false-color (= pseudocolor) images to facilitate area integration [22] (Fig. 1). Generally there is a list of geometric, densitometric, and textural parameters that could be selected by the researcher on the basis of his/her needs. Some image analyzers also allow researchers to construct parameters of their choice [22]. The most common features are nuclear area, nuclear absorbing area, nuclear perimeter, roundness, mean gray value per nucleus (which may be converted into absorbance (optical density, OD)), absorbance variability per nucleus, contrast, entropy, energy, minimum feret, maximal feret, feret ratio, and many others.
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A feret diameter is the distance between two parallel lines drawn tangentially to opposite sides of an object boundary [23]. The maximal feret corresponds to the largest long axis of the object; the minimal feret corresponds to the shortest width axis perpendicular to the largest long axis of the object [23]. The feret ratio (minimal feret/maximal feret) is an indicator of the elongation of the absorbing object. The integrated OD (= IOD) is equal to the absorbance multiplied by the nuclear absorbing area. For Feulgen-stained nuclei, IOD represents Feulgen-DNA values in arbitrary units (see Notes 10 and 11).
4
Notes 1. An absolute ethanol-glacial acetic acid mixture is the best fixative for DNA studies that utilize the Feulgen reaction. 2. In our laboratory, we ordinarily use 4 M HCl at room temperature for ethanol-acetic acid-fixed materials. Because the hydrolysis time varies according to the acid concentration and temperature as well as the material specificities, time course should be performed to determine the time that is mostly adequate for maximal DNA depurination, that is, that which provides the largest staining intensity. 3. Paraffin sections may be used for establishing Feulgen-DNA values provided the sections are thick enough to contain whole nuclei for measurements. If hematoxylin-stained sections are used to study the degree of compactness and the distribution of chromatin condensed areas, special care should be taken and a larger sample size should be used. 4. It is recommended that the microspectrophotometer construction characteristics reduce glare effects and diffraction errors. 5. We use Planapo objectives 63/0.90 or 40/0.95, optovar 2.0, measuring diaphragm 0.25 mm, and LD-Epiplan 16/0.32 condenser to analyze nuclei from mammalian hepatocytes, cardiomyocytes, mammalian culture cells, and nucleated erythrocytes. An R-928 photomultiplier is a component of a Zeiss automatic scanning microspectrophotometer. 6. In our experience, an interference filter of λ = 546 nm is also adequate for measuring the Feulgen-stained response if the interference filter of λ = 565 nm is not available. 7. We use Neofluar or UPlanF1 objectives 40/0.75 and optovar 2.0 for nuclei from mammalian hepatocytes, mammalian culture cells, nucleated erythrocytes, and insect cells.
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8. The Feulgen reaction should not be followed by any treatment with ethanol solutions; otherwise, the staining will become partially removed. 9. Sometimes, more than one cutoff point can be selected (e.g., 0.100, 0.200) to obtain results with a better capacity for envisioning different chromatin condensation degrees [1, 14, 22]. 10. Arbitrary units can be converted into absolute units (e.g., DNA picograms, 10−12 g) if a control with a known DNA concentration is used. 11. To study the changes in chromatin packing states, the best features for comparisons are absorbance variability per nucleus, contrast, entropy, and energy.
Acknowledgments This work was supported by the São Paulo State Research Foundation (FAPESP, grants no. 2010/50015-6 and 2009/11763-0) and the Brazilian National Council on Development and Research (CNPq). References 1. Mello MLS, Contente S, Vidal BC et al (1995) Modulation of ras transformation affecting chromatin supraorganization as assessed by image analysis. Exp Cell Res 220:374–382 2. Vidal BC, Russo J, Mello MLS (1998) DNA content and chromatin texture of benzo[a] pyrene-transformed human breast epithelial cells as assessed by image analysis. Exp Cell Res 244:77–82 3. Gilbert N, Gilchrist S, Bickmore WA (2005) Chromatin organization in the mammalian nucleus. Int Rev Cytol 242:283–336 4. Cremer T, Zakhartchenko V (2011) Nuclear architecture in developmental biology and cell specialization. Reprod Fertil Dev 23:94–114 5. Felisbino MB, Tamashiro WMSC, Mello MLS (2011) Chromatin remodeling, cell proliferation and cell death in valproic acid-treated HeLa cells. PLoS One 6:e29144 6. Felisbino MB (2012) Remodelação cromatínica, anomalias cromossômicas e morte celular em condições de inibição de deacetilases de histonas em células HeLa e NIH 3T3. Masters’ dissertation, University of Campinas, Campinas, p 106 7. Tóth KF, Knoch TA, Wachsmuth M et al (2004) Trichostatin A-induced histone acetylation
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