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Method for detecting nucleoproteins of spermatogenic cells through utilizing indirect immunofluorescent staining

A technology of immunofluorescence staining and cell nucleus, which is applied in the field of detection of nuclear protein in male mammalian spermatogenic cells, can solve the problems of protein loss in the nucleus, achieve the effects of inhibiting degradation, good water solubility and penetration, and promoting permeability

Inactive Publication Date: 2013-08-07
SHANXI MEDICAL UNIV
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Problems solved by technology

However, high concentrations of TitonX-100 are more destructive to biofilms, and can dissolve cells and their internal organelles, which may cause loss of nuclear proteins

Method used

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Embodiment Construction

[0022] Taking mice as an example, the operation steps of the method for detecting spermatogenic nuclear protein by indirect immunofluorescence staining of the present invention will be described in detail below. However, the method of the present invention is not limited to mice, and the spermatogenic cells of other male mammals can also use the method of the present invention for indirect immunofluorescent staining of nucleoproteins.

[0023] 1. Execute male mice of appropriate age by neck dislocation, soak in 75% ethanol, take out the mouse testis tissue aseptically, and place them in a plate containing pre-cooled PBS solution.

[0024] 2. Carefully peel off the buffy coat, remove blood vessels as much as possible, fully separate the seminiferous tubules, and transfer to another plate containing pre-cooled PBS solution.

[0025] 3. Wash several times with PBS solution to remove other impurities such as tissue fragments.

[0026] 4. Under a dissecting microscope, stretch the...

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Abstract

A method for detecting the nucleoproteins of spermatogenic cells through utilizing indirect immunofluorescent staining comprises the following steps: separating convoluted seminiferous tubules from the testis tissues of an animal, utilizing a hypotonic solution to process spermatogenic cells, and detecting the nucleoproteins of the spermatogenic cells through adopting indirect immunofluorescent staining, wherein the hypotonic solution comprises 17mM of sodium citrate, 50mM of sucrose, 5mM of EDTA, 0.5mM of DTT, 0.5mM of PMSF and 30mM of Tris and having a pH value of 8.2. The method uses the hypotonic solution to carry out permeable treatment of the cells in order to rapidly expand the cells, disperse chromatins and fully release the proteins in cell nuclei, so the spermatogenic cells can well combine with primary antibodies, thereby the combination efficiencies of secondary antibodies are enhanced, fluorescent staining is complete and full, and the preparation of spermatogenic cell chromosomes and karyotyping are successfully carried out.

Description

technical field [0001] The invention relates to a biochemical detection method, in particular to a detection method of male mammalian spermatogenic nucleoprotein. Background technique [0002] At present, the incidence of infertility has been as high as 15% to 20%, of which the male factor accounts for about one-half. As the main problem of male reproductive health, male infertility is related to the overall quality of the nation and the healthy development of the future population. It has become a focus issue that urgently needs social attention and needs to be resolved. Carrying out basic research on male reproductive health and revealing the molecular mechanism of spermatogenesis and maturation can provide a laboratory basis for elucidating the pathogenesis of male infertility and contribute to the development of clinical diagnostic techniques for male infertility. [0003] Mammalian spermatogenesis is a complex and specific cell differentiation process, which is divided...

Claims

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Application Information

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IPC IPC(8): G01N33/68
Inventor 郭睿闫萍于保锋梁样红杨丽娟张小曼刘丹李春锋高然朋解军
Owner SHANXI MEDICAL UNIV
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