Chromosomal gains or losses were analyzed using standard CGH techniques. DNA derived from U-DCS cells was Nick-translated and labelled with a red fluorescing dye (Cy5). Then, control metaphase chromosomes were co-incubated with the red fluorescing cell line DNA and green fluorescing control DNA (Abbott, Wiesbaden, Germany) and analyzed using an Axioplan 2 microscope (Zeiss; Jena, Germany). Gains and losses were identified by comparing the green and red fluorescence signals (ISIS3; MetaSystems; Altlussheim, Germany).
Isis3
Manufactured by MetaSystems
Sourced in Germany
ISIS3 software is a data processing and analysis tool designed for the scientific community. It provides a comprehensive suite of capabilities for handling and interpreting data from various sources, including remote sensing and laboratory instruments. The software's core function is to enable users to perform advanced data analysis, visualization, and modeling tasks in a versatile and user-friendly environment.
Automatically generated - may contain errors
Lab products found in correlation
Coolcube ccd camera, by MetaSystems (2 mentions)
Bx60 fluorescence microscope, by Olympus (2 mentions)
Metafer slide scanning system, by MetaSystems (2 mentions)
Axioplan 2 microscope, by Zeiss (1 mentions)
Avidin fitc, by Vector Laboratories (1 mentions)
Anti digoxigenin rhodamine, by Roche (1 mentions)
Mib 1, by Agilent Technologies (1 mentions)
Cy3 conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Alexa fluor 488 kit, by Thermo Fisher Scientific (1 mentions)
Nick translation reagent kit, by Abbott (1 mentions)
Green dutp, by Abbott (1 mentions)
7 protocols using isis3
1
Chromosomal Gain/Loss Analysis using CGH
2
FISH Metaphase Spread Preparation
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Metaphase spreads for FISH (fluorescence in situ hybridization) analysis were prepared from lymphocytes or fibroblast cell cultures using standard methanol:acetic acid (3:1) fixation. Fluorescent probes were hybridized as previously described [27 (link)]. Slides were washed in 0.4 × SSC/0.3% Igepal at 73 °C, counterstained and mounted with DAPI/Antifade. The FISH preparations were evaluated using Olympus BX 51 and BX 60 microscopes equipped with the necessary fluorescence filters and automated pad shifts. Good quality metaphase cells were scanned using a CCD camera and evaluated using image analysis (ISIS 3, MetaSystems, Altlussheim, Germany).
3
Comparative FISH Analysis of Cervine and Capreoline Satellite DNA
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Probes for satI, satII, satIII-partial, and satIV of C. elaphus (Cervinae) and satI-IV of R. tarandus (Capreolinae) were labelled with orange—or green—dUTP and used for comparative FISH. Moreover, we used satI (NCBI accession numbers: V00124 and Z18540) and satII (NCBI accession numbers: M36668 and AF245169) probes derived from two bovid species (B. taurus and O. aries). The FISH was carried out according to standard protocols [26 (link)]. Hybridisation signals were examined using an Olympus BX60 fluorescence microscope equipped with appropriate fluorescent filters. Images of well-spread metaphase cells were captured by a CoolCube CCD camera and analysed using ISIS3 software (version 5.8.3, MetaSystems, Altlussheim, Germany).
4
Fluorescence in situ Hybridization of Sperm
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FISH and sperm-FISH procedures were carried out as described in Vozdova et al. 2019 [32 (link)]. BAC probes labeled with digoxigenin-11-dUTP were detected with antidigoxigenin rhodamine (Roche Diagnostics, Indianapolis, IN, USA). BAC probes labeled with biotin-16-dUTP were detected with Avidin-FITC (Vector Laboratories, Inc., Burlingame, CA, USA). Hybridization signals were examined using Zeiss Axio Imager.Z2 fluorescence microscope (Carl Zeiss Microimaging GmbH, Jena Germany) equipped with appropriate fluorescent filters and the Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). Images of well-spread metaphase cells were captured and analyzed using ISIS3 software (MetaSystems, Altlussheim, Germany).
5
Immunohistochemical Analysis of CARD9 and Cell Markers
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Immunochemistry was performed on formalin-fixed, paraffin-embedded (FFPE) tissue and cell pellets using the avidin–biotin complex method with the AP/RED Detection System (K5005, Dako, Hamburg, Germany). The following antibodies were used: rabbit anti-CARD9, 1:100 (C7862, Sigma-Aldrich, St. Louis, MO, USA); mouse CD20, 1:500 (L26, Dako); and mouse anti-KI67, 1:200 (MIB1, Dako). For immunofluorescence double staining, Cy3-conjugated goat anti-mouse IgG, 1:400 (Jackson Immunoresearch, Westgrove, PA, USA) was used to detect the CD20 antibody; and biotin-conjugated pig anti-rabbit IgG, 1:200 (DAKO) followed by signal amplification with the Alexa Fluor-488 kit, 1:1600 (Thermo Fisher, Waltham, MA, USA) detected the anti-CARD9 antibody. Images were processed using ISIS3 software (Metasystems, Heidelberg, Germany).
6
Fluorescence In Situ Hybridization of Deer Satellite DNA
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Cloned satI, satII, satIII, and satIV DNA of M. gouazoubira were labelled with Orange- or Green-dUTP (Abbott, Abbott Park, IL, USA) using Nick Translation Reagent Kit (Abbott) to serve as probes for comparative FISH. FISH was performed using standard protocols [19 (link)]. Hybridization signals were examined using Zeiss Axio Imager.Z2 fluorescence microscope (Carl Zeiss Microimaging GmbH, Jena, Germany) equipped with appropriate fluorescent filters and the Metafer Slide Scanning System (MetaSystems, Altlussheim, Germany). Images of well-spread metaphase cells were captured and analyzed using ISIS3 software (MetaSystems).
7
Quantifying Meiotic Recombination Sites
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For the analysis, an Olympus BX60 fluorescence microscope equipped with appropriate fluorescent filters was used. Images of well-spread pachytene spermatocytes with MLH1 signals were captured by a CoolCube CCD camera (MetaSystems, Altlussheim, Germany). The number of MLH1 foci (recombination sites) per cell, distance of MLH1 foci from the centromere (μm) and all SC lengths (μm) were scored using Isis3 software (MetaSystems).
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