Pig oocyte membrane protein NHE1 fluorescent staining method

An oocyte, NHE1 technology, applied in the field of cell biology to avoid interference

Pending Publication Date: 2021-02-02
INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the position of the membrane protein NHE1 (sodium / hydrogen exchange factor 1) close to the zona pellucida, the presence of the zona pellucida often prevents the cell membrane protein from specifically binding to the specific antibody or non-specifically binding in the fluorescent staining experiment. Observation of effects on the zona pellucida

Method used

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  • Pig oocyte membrane protein NHE1 fluorescent staining method
  • Pig oocyte membrane protein NHE1 fluorescent staining method
  • Pig oocyte membrane protein NHE1 fluorescent staining method

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0033] Embodiment 1. The method for fluorescent staining of porcine oocyte membrane protein NHE1 comprises the steps:

[0034] 1) Porcine oocyte culture: Porcine ovaries are taken from sexually mature young sows in local slaughterhouses, and the ovaries are stored in 25-30°C normal saline containing penicillin and streptomycin and sent back to the laboratory as soon as possible. Cumulus-oocyte complexes (COCs) were withdrawn from medium-diameter (2-6 mm) follicles using a 10 mL disposable syringe fitted with a pink needle. Under the stereoscope, select oocytes with tightly packed cumulus cells and evenly distributed cytoplasmic granules, rinse them in the mature culture medium for several times, and then transfer them into the mature culture medium. Cultured in a humidified incubator and collected at 0, 24, 30 and 44 hours of in vitro maturation, germinal vesicular phase (GV), first mature prophase (GVBD), first mature division metaphase (MI), second mature division Metaphase...

Embodiment 2

[0043] Example 2. Repeat Example 1, the difference from Example 1 is that the membrane penetration time at 37°C in step 3) in this example is 48h.

Embodiment 3

[0044] Example 3. Repeat Example 1, the difference from Example 1 is that the membrane penetration time at 37°C in step 3) in this example is 8h.

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Abstract

The invention discloses a pig oocyte membrane protein NHE1 fluorescent staining method, and relates to the technical field of cell biology. In order to more visually observe the period of cells and the co-localization condition of membrane protein NHE1 and microfilaments, the invention provides a porcine oocyte membrane protein NHE1 fluorescent staining method which comprises the following steps:extracting a cumulus oocyte complex from sexually mature porcine follicles, culturing, obtaining oocytes in a foaming period GV, a primary maturation and division early-stage GVBD, a primary maturation and division medium-stage MI and a secondary maturation and division medium-stage MII, sequentially removing cumulus cells through hyaluronidase and removing zona pellucida through streptavidin, performing membrane permeation and sealing on pig oocytes in each period, performing antibody incubation, and dyeing nuclei to prepare a sealing sheet, and observing under a multichannel confocal microscope. The invention provides a new technical support for researching the molecular mechanism of the porcine oocyte membrane protein NHE1 and other membrane proteins.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for fluorescent staining of porcine oocyte membrane protein NHE1. Background technique [0002] The maturation process of porcine oocytes involves many different developmental stages, and its fluorescent staining can visually display the different stages of porcine oocyte development. Due to the position of the membrane protein NHE1 (sodium / hydrogen exchange factor 1) close to the zona pellucida, the presence of the zona pellucida often prevents the cell membrane protein from specifically binding to the specific antibody or non-specifically binding in the fluorescent staining experiment. Observation is affected by the zona pellucida. Contents of the invention [0003] In order to more intuitively observe the stage of the cells and the colocalization of the membrane protein NHE1 and microfilaments, the invention provides a method for fluorescent staining of the po...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/30
CPCG01N1/30
Inventor 王丽英许保增张颖赵伟刚杨镒峰任雨贺李晓霞刁云飞王士勇韩玉萍李文
Owner INST OF SPECIAL ANIMAL & PLANT SCI OF CAAS
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