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    Home > Biochemistry News > Biotechnology News > The dyeing system of bone marrow in mice is prepared.

    The dyeing system of bone marrow in mice is prepared.

    • Last Update: 2020-10-25
    • Source: Internet
    • Author: User
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    . 1.
    Experimental Principles Mice bone marrow cells have a high degree of division activity and are in large numbers, so there is no need for in-body
    culture
    to directly observe the division phase of cells. Cells in the division phase are treated with akiylasin, which blocks the formation of spindle wire and stops cell division in the medium term, when chromosomes reach maximum contraction and have a typical form. Low seepage processing breaks cells and allows chromosomes to disperse. This method is used in the clinical study of leukemia, and can also be used to observe the toxic substances on the body's genetic material - chromosomal damage.
    mouse chromosomes
    2n-40,
    were all
    ,
    males with silk chromosomes
    40
    ,
    XY
    , females
    40
    ,
    XX
    .
    2.
    . Experiments
    reagents
    and equipment
    microscope
    ,
    ringer
    water bath pot, low speed
    centrifuge
    , electronic
    balance
    , anatomical instruments (enamel anatomical disc, scissors, tweezers), syringes, needles,
    test tubes
    , test tube racks, droppers, load glass.
    0.85
    % physiological saline, fixed liquid
    (
    methanol: ice acetic acid is
    3
    :
    1)
    ,
    PBS (pH6.8)
    ,
    Giemsa
    Liquid, akilets
    (
    dissolved in
    0.85
    % physiological saline
    ) at a concentration of
    100
    μ
    g/ml
    )
    ,
    0.075mol/L KCl
    .
    3.
    . Experimental methods and steps
    (
    1
    ) autumn daffodil treatment:
    3h
    before taking bone marrow, the mouse abdominal cavity was injected with autumn daffodils at a dose of
    100
    μ
    g
    /
    kg
    animal weight.
    (
    2
    ) Take bone marrow: Kill the mice with spinal cord injury, then use scissors to cut open the skin and muscles on the thighs, remove the thigh bone, and clean the muscle residue attached to the bone with a small piece of gauze. Cut off the puffed joint heads at both ends of the collarbone, then use a syringe to absorb
    5 ml 0.85
    % physiological saline, insert it into one end of the collarbone, and rinse the bone marrow cells into the
    centrifuge tube
    of
    10 ml
    . Rinse repeatedly until the bone marrow cavity is white.
    (
    3
    ) low seepage treatment: the resulting cell suspension at
    1000rpm
    centrifugation
    10min
    , absorb the liquid, leave
    0.2ml
    sediment, plus
    0.075mol/L KCL
    solution (
    37
    degrees C) to
    8 ml
    scale, the cell sediment blown apart well, in
    37
    degrees C water bath low seepage
    25min
    .
    (
    4
    ) fixed: after low seepage treatment, immediately add
    1 ml
    newly provisioned and pre-cooled fixation liquid, pump evenly, and then
    1000 rpm
    centrifugation
    10min
    , discarded. Add the fixation fluid along the pipe wall to
    6 ml
    , and stationary fixation
    10min
    after blowing away the cells, i.e. for the first time. According to the above conditions centrifugation, go to the liquid, add the fixing liquid again to
    6 ml
    , for a second fixation.
    10min
    centrifuged to suck up the liquid, leave
    0.2ml
    sediment, and then add
    4
    drops of fixation to the sediment, flushed to make cell suspension.
    (
    5
    ) drops: take the pre-cooled slides in the refrigerator, from about
    15cm
    high to each slide drops
    1
    to
    2
    drops of cell suspension on the adhesive ice water slide, drying.
    (
    6
    ) staining: take the film flat on the glass plate, with
    1
    :
    9 Giemsa
    dye dye
    20min
    , water rinsed and dried.(
    7
    ) observed the morphological characteristics of the chromosomes of mice and calculated the number of chromosomes
    2n
    of chromosomes.
    .
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