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22 protocols using pararosaniline

1

Histologic Processing and Analysis of Tissue Samples

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Histologic processing was done as described [15 (link)]. The specimen was dehydrated in 100 % ethanol and subsequently infiltrated, embedded and polymerized in Technovit 9100 (Heraeus Kulzer, Wehrheim, Germany) according to the manufacturer’s instructions. After the polymerization process, samples were cut into 500 μm sections using a rotary diamond saw Secotom- 50 (Stuers, Ballerup, Denmark). The sections were mounted onto opac acrylic-slides (Maertin, Freiburg, Germany) and ground to a final thickness of approximately 60 μm on a rotating grinding plate (Stuers, Ballerup, Denmark). All specimens were stained with azure II and pararosaniline (Merck, Darmstadt, Germany).
Slides were imaged with an Axio Imager M1 microscope equipped with a digital AxioCam HRc (Carl Zeiss, Göttingen, Germany). The histologic sections were analyzed via analySIS FIVE – software (Soft Imaging System, Münster, Germany).
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2

Microscopic Analysis Reagent Preparation

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The chemicals used in this study were purchased from the following suppliers: activated coal, ethanol, fast green, HCl, methanol, pararosaniline, sodium bisulfite, and xylene from Merck Chemicals (Darmstadt, Germany), water bath from Isolab (Eschau, Germany), and light microscope from Olympus (Tokyo, Japan).
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3

Histological Analysis of Tibial Osteoclasts

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The tibia samples were decalcified in EDTA (BDH Chemicals) and processed into paraffin blocks (Tek III Paraffin Wax, Sakura, Netherlands). Midsagittal 4 μm FFPE sections were obtained from each sample. The sections were stained with hematoxylin and eosin and Orange G (HE-Orange G, reagents from Sigma-Aldrich and Acros Organics) for basic histological evaluation of the tumor and bone and with pararosaniline (Sigma-Aldrich) for the staining of osteoclasts using standard methods. The stained sections were scanned with a digital slide scanner (Pannoramic Scanner 250, 3DHISTECH) and analyzed with the Pannoramic Viewer and HistoQuant (3DHISTECH). Tumor and bone areas were defined in each section from the growth plate to 5 mm distance to the distal tibia and analyzed by color-thresholding. Also, the number of osteoblasts and osteoclasts was analyzed from these images.
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4

Mitotic Index Analysis in Plant Root Tips

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For each treatment condition, 1.0 cm long sections from 20 roots were isolated, washed in ddH2O and fixed overnight in ethanol:glacial acetic acid (v: v = 3:1) at 4 °C before successively rinsing for 10 min with 100% ethanol, 95% ethanol and 75% ethanol. The treated root sections were stored at −20 °C in 70% ethanol. The root-tips were cut into 1.0–1.5 mm sections and transferred to enzyme mixtures containing 4% cellulose (Sigma), 1% pectolyase (Sigma), and 0.5% pectinase (Sigma) and incubated at 37 °C for 1 h. The root tips were then washed in ddH2O, hydrolyzed in 5 M HCl for 20 min at room temperature, washed three times in ddH2O, and stained with Schiff’s reagent (pararosaniline; Sigma-Aldrich) for 1 h at room temperature. To prepare slides for observation of cells, 10 uniform root tips were selected. More than 1,000 cells per slide were randomly selected and photographed using an AxioCam MRc5 and AxioVision v. 4.7 software (Carl Zeiss Microscope, Gottingen, Germany). The average mitotic index was estimated based on 30 images with at least 3000 cells. The mitotic index data were analyzed and plotted using Origin 9.1. Mitoticindex=TotalnumberofmitoticcellsperimageTotalnumberofcellsperimage
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5

Quantitative Feulgen DNA Analysis

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Apical root fragments were fixed for 60 min in cold Carnoy’s mixture (absolute ethanol and glacial acetic acid; 3:1, v/v). After washing with ethanol (3 times), root tips were rehydrated, hydrolysed for 1 h in 4M HCl, and Feulgen-DNA-stained using the standard method [46 ] with pararosaniline (applied to selectively stain DNA in a quantitative colorimetric measurement of aldehydes in the Schiff’s test; Sigma-Aldrich, Poznan, Poland). Following 3 times rinsing with SO2 water (sulphurous acid prepared from sodium metabisulphite and dilute HCl to stop Feulgen staining, to fix the color, and to elute unbound molecules) and after washing with distilled water, root tips were crushed on microscope slides in 45% acetic acid. Squash preparations were frozen on dry ice, dried, and embedded in Canadian balsam. The total number of cells analysed was always 8000 (out of 10 root meristems) at each time point. Selected cells were photographed with E-600 epifluorescence microscope (Nikon). The extinction of Feulgen-stained cell nuclei was measured at 565 nm with a Jenamed 2 microscope (Carl Zeiss, Jena, Germany) and calibrated in arbitrary units (a.u.). A computer system (Forel, Łódź, Poland) was used for image analysis. Approximately 5000 nuclei were collected to evaluate the distribution of the DNA content.
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6

TRAP Staining for Osteoclast Differentiation

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After 3, 7 and 14 days of culture, cells were washed with PBS, fixed with 4% PFA (Sigma-Aldrich, France) for 10 min at RT, then tartrate-resistant acid phosphatase (TRAP) activity was detected by enzyme histochemistry to assess OC differentiation. Briefly, cells were stained for acid phosphatase, using naphthol ASTR phosphate (Sigma) as a substrate in the presence of 50 mM tartrate and hexazotized pararosaniline (Sigma), and counterstained with methyl green (Sigma). TRAP positive cells were counted under microscope.
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7

Caco-2 Cell Colony Formation Assay

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Determination for colony formation was assessed by seeding the Caco-2 (p. 50) in cultures with SPF into six-well plate. The cells were cultured in CGM and the cell density was equal to 100 cells per well. Next, the cells were maintained for 10 days in a CO2 incubator (5% CO2 and 95% humidity). The medium was removed and cells were fixed with 4% paraformaldehyde (PFA) and stained with pararosaniline (Sigma Aldrich) for 10 min at room temperature. The CFU were counted as colonies consisting only of 50 or more cells according to the method described previously [24 (link)]. The experiment was performed three times.
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8

Wound Healing Assay in Caco-2 Cells

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In order to perform the wound healing test, the Caco-2 cultures (p. 50) were maintained in 24-well plates, enforcing high confluency. Cell monolayers were manually wounded by scraping cultures with yellow pipette tips (0.5–0.6 mm diameter). After that, cultures were washed twice to remove the detached cells and propagated with SPF in a CO2 incubator for 24 h. Furthermore, cultures were stained with pararosaniline (Sigma Aldrich, Munich, Germany). Determination of wound closure was performed using AxioVision microscope software. The measurements of distance, obtained between the two edges of a denuded area, were performed five times and repeated independently in triplicate.
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9

Cell Cycle Analysis Reagents

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Hydroxyurea (HU, 2.5 mM), sodium metavanadate (Van, 200 µM), pararosaniline, N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), bovine serum albumin (BSA), polyvinylpyrrolidone (PVP-40), propidium iodide (PI), and 4′,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma. Caffeine (CF, 5 mM) was supplied by Merck, Triton X-100 and pectinase from Aspergillus niger by Fluka, cellulase Onozuka R-10 from Trichoderma viride, and RNase from SERVA. 2-aminopurine (2-AP, 10 mM) and pectolyase Y-23 were obtained from ICN Biomedicals. Other chemicals were obtained from POCH S.A.
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10

Quantifying Nuclear DNA Content

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Apical fragments of embryo roots and cotyledons were fixed in cold Carnoy’s mixture (glacial acetic acid and absolute ethanol; 1:3; v/v) for 1 h. Following rehydration (70% ethanol, 30% ethanol, distilled water), the roots were hydrolyzed in 4 M HCl for 1 h and stained with Schiff’s reagent (pararosaniline; Sigma-Aldrich, St. Louis, MO, USA) according to the standard methods [83 (link)]. After rinsing in SO2-water and then in distilled water, fragments of cotyledons from the selected zones and 1.5-mm-long apical segments of the roots were cut off and squashed onto Super-Frost (Menzel-Gläser, Braunschweig, Germany) microscope slides. Following freezing with dry ice, cover slips were removed, and the dehydrated dry slides were embedded in Canada balsam. Nuclear DNA content was evaluated by means of microdensitometry using a Jenamed 2 microscope (Carl Zeiss, Jena, Germany) with the computer-aided Cytophotometer v1.2 (Forel, Lodz, Poland). The Feulgen-stained cell nuclei were measured at 565 nm. Microscopic slides were used also to analyze the mitotic and phase indexes.
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