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Equipment and applied methods G&EB

Institute of Animal Reproduction and Food Research of PAS > Research & Dissemination > Scientific Groups > Gamete and Embryo Biology > Equipment and applied methods G&EB

Embryo Biology Team

Laboratory methods

    1. Methods in vitro:
      • Enzymatic isolation and culture of the reproductive tract cells,
      • Incubation and culture of tissues of reproductive tract of the cow,
      • in vitro embryo culture (time laps system as well),
      • Diagnostic biopsies of trofectoderm,
      • siRNA microinjection into oocytes and embryos,
      • Intracytoplasmic morphologically selected sperm injection (IMSI),
      • Immunofluorescent staining of oocytes and embryos,
      • Embryo/gamete vitrification.
    2. Analytical methods:

2.1. Molecular biology methods:

    • Nucleic acid isolation (from small amount of genetic material)
    • Reverse transcription
    • Analysis of mRNA expression of various factors by methods of conventional PCR and quantitative real time PCR using SYBR Green and TaqMan probes .
    • Protein level examination by Western-blotting method.

2.2. Determination of protein and steroid hormones, and prostaglandins with immunoenzymatic (ELISA) method with hormones labeled with horseradish peroxidase (HRP) or biotin.

2.3. Immunohistochemistry and immunofluorescence staining for selected substances in the cells and tissues of reproductive tract and embryos.

Equipment

  1. Oocyte isolation and in vitro embryo culture workplaces (3):
    • Stereomicroscope ZEISS Stereo Discovery v. 20 (2 pcs.)
    • Stereomicroscope Olympus SZX7
    • Stereomicroscope Olympus SZX10
    • Incubators Esco Miri® Multi-Room Incubator, EscoMedical (3 pcs.)
    • Multi gas incubator with CO2-N2/O2 atmosphere Panasonic MCO-5M-PE
    • Incubator Memmert IN30
    • Incubator IB-05 G
    • Msc-advantage Biological Safety Cabinet with flow laminar, ALAB
    • Autoklave Speedyclave 6B, Cominox
    • Centrifuge 5804R with fan cooling, Eppendorf
    • Centrifuge Minispin, Eppendorf
    • Thermo plates, Semic Bioelectronika
    • Thermo plates MATS-U4020-WF, Olympus
    • Thermo plates HT50, Minitube
    • Thermoblocks, PeqLab
    • Incubator for tissues transporting, Semic (3 szt.)
  2. Gamete and embryo micromanipulation workplace:
    • Micromanipulator Eppendorf Transfer Man NK2 with stereomicroscope Zeiss AXIO Observer.A1
    • Origio Fortuna IVF Clean Air Cabinet with flow laminar, with stereomicroscope Zeiss Discovery.v20
    • Micropipette puller P-1000, Sutter Instrument
    • Micro Grinder Narishige EG-400
    • Micro Forge Narishige MF-900
  3. Primo Vision System
    • Three-gas incubator MCO-19M-PE, Panasonic
    • Primo Vision System
  4. Cell and tissue culture workplace:
    • Msc-advantage Biological Safety Cabinet with flow laminar, ALAB
    • Incubator Memmert IN30 30-750
    • Incubator with shaker IKA® KS4000 iControl
    • Incubator CO2/O2  MCO-18M, Sanyo
    • Incubator MCO-5M-PE, Panasonic
  5. Nucleic acids isolation workplace:
    • Thermoblock Digital Dry Bath, BioRad
    • Vortex, Heidolph Reax Top
    • Centrifuge Super Mini Centrufuge Mini-14K, Hangzhou Ausheng Instruments
    • Incubator Major Science  MO-01-220
  6. Western blot i immunofluorescence workplace
    • Thermoclock ALB64
    • Centrifuge Minispin, Eppendorf
    • Laboratory scale WPS110/C/Z, Radwag

Semen Biology Team

Molecular Andrology Team

  1. Fast protein liquid chromatography system (FPLC system)
    Isolation of proteins and peptides from vertebrate sperm, determination of the main physico-chemical properties of proteins.
  2. High-performance liquid chromatography system (HPLC system)
    Isolation of proteins and peptides, separation of peptides obtained after protein digestion, preparation of products for sequencing and mass spectrometry.
  3. Spectrophotometer Beckman DU 640, portable spectrophotometer Eppendorf
    Measurement of sperm concentration, an indication of the basic biochemical determinants of semen quality, measurement of antioxidant activity of the semen, kinetic analysis (determination of enzyme and inhibitor concentration, measurement of the equilibrium association constant for inhibitor – proteinase).
  4. Apparatus for measuring sperm concentration and viability (NucleoCounter SP-100)
    Measurement of sperm concentration and viability.
  5. An integrated system for the comet assay
    Assessment of sperm genome.
  6. Set vertical electrophoresis
    Visualization of seminal plasma protein profiles.
  7. Apparatus for transfer
    Transfer of proteins on the membrane, Western blot analysis.
  8. Osmometer
    Measurement of seminal plasma osmotic pressure.
  9. Additional equipment
    • Centrifuge with cooling,
    • Ultra low temperature freezer.