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3D cell microsphere paraffin sectioning method

A technology of paraffin sectioning and microspheres, applied in sampling, measuring devices, instruments, etc., can solve the problems of insufficient wax soaking and difficult sectioning, etc.

Inactive Publication Date: 2021-05-14
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The agarose embedding method commonly used for paraffin sections of cell balls may be affected by the concentration of agarose and the melting state, which may result in insufficient wax immersion and difficulty in slicing.

Method used

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  • 3D cell microsphere paraffin sectioning method
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  • 3D cell microsphere paraffin sectioning method

Examples

Experimental program
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Effect test

Embodiment 1

[0050] Embodiment 1 HE stained human hepatic stellate cell (LX-2) cell microsphere paraffin section

[0051] In this embodiment, paraffin sections of LX-2 cell microspheres were prepared according to the method of the present invention, and the sections were stained with HE. The specific steps are as follows:

[0052] 1. Sample fixation: Collect the cultured 3D hepatocyte microspheres into a centrifuge tube. After the cell spheres are sedimented or centrifuged at low speed to the bottom of the centrifuge tube, remove the supernatant medium and add an appropriate amount of 10% neutral formalin , fixed at room temperature for 30 minutes;

[0053] 2. Pre-staining: add water-soluble eosin dye solution and dye for 2 minutes;

[0054] 3. Dehydration: Remove the eosin dye solution and add gradient ethanol or its solution for gradient dehydration, followed by ① 75% ethanol for 5 minutes, ② 90% ethanol for 5 minutes, ③ 95% ethanol for 3 minutes, ④ absolute ethanol for 3 consecutive ti...

Embodiment 2

[0075] Example 2 Paraffin sections of HepG2 and LX-2 cells co-cultured with immunohistochemical staining

[0076] This embodiment mainly uses the method of the present invention to prepare paraffin sections of HepG2 and LX-2 cell co-cultured cell microspheres, wherein the steps from sample fixation to rehydration are the same as in Example 1, and immunohistochemical staining is used for the sections. The specific dyeing steps are as follows:

[0077] 1. Antigen retrieval: put the rack with slices into PBS (0.01M, pH 7.2-7.4) for cleaning, and microwave Tris / EDTA antigen retrieval solution (10mM Tris, 1mM EDTA, 0.05% Tween-20, pH 9.0 ) to 100°C, put the slide rack with slices into the boiling Tris / EDTA repair solution, heat on medium heat for 20 minutes, take out the container and place it in ice water for rapid cooling for about 10 minutes.

[0078] 2. Permeabilization: Incubate the sample in 1x TBS (pH 7.6) containing 0.025% Triton X-100 for 10 minutes, wash with PBS 3 times...

Embodiment 3

[0101] Example 3 Immunofluorescent staining of HepG2 and LX-2 cells co-cultured cell microsphere paraffin section

[0102] In this example, paraffin sections of co-cultured cell microspheres of HepG2 and LX-2 cells were prepared according to the method of the present invention, wherein the steps from sample fixation to rehydration were the same as in Example 1, and the sections were stained with immunofluorescence, and the details of the staining Proceed as follows:

[0103] 1. Antigen retrieval: put the rack with slices into PBS (0.01M, pH 7.2-7.4) for cleaning, and microwave Tris / EDTA antigen retrieval solution (10mM Tris, 1mM EDTA, 0.05% Tween-20, pH 9.0 ) to 100°C, put the slide rack with slices into the boiling Tris / EDTA repair solution, heat on medium heat for 20 minutes, take out the container and place it in ice water for rapid cooling for about 10 minutes.

[0104] 2. Permeabilization: Incubate the sample in 1x TBS (pH 7.6) containing 0.025% Triton X-100 for 10 minut...

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Abstract

The invention relates to a 3D culture cell microsphere paraffin sectioning method, which comprises the steps of sample fixation, pre-dyeing, dehydration, transparentizing, paraffin dipping and embedding, sectioning, slice fishing, slice unfolding, slice baking, dewaxing, rehydration, dyeing and the like, and is characterized in that the sectioning method is simple, convenient and rapid, and only about 2 hours are needed from collection of cell spheres to preparation of paraffin blocks. According to the method, the section structure prepared from the 3D cell microspheres with the diameter of 50-1000 microns is complete, the cell boundary is clear, the subsequent operability and practicability are high, and an effective means is provided for researching the internal characteristics of the 3D cell spheres.

Description

technical field [0001] The invention relates to a paraffin sectioning method, in particular to a 3D cell microsphere paraffin sectioning method. Background technique [0002] Compared with 2D cell culture, 3D cell culture can provide cells with a microenvironment that is closer to the living conditions in vivo, better simulate the physiological state, and obtain experimental results that are more consistent with in vivo experiments. With improvements in biological relevance, ease of use, and throughput of some new 3D cell culture technologies, 3D cell culture is increasingly used in basic research and drug discovery. Cell microspheres are a simple in vitro 3D cell culture model, common ones include embryonic bodies, mammospheres, tumor spheres, hepatocyte spheres, neurospheres, etc. Compared with more complex 3D models, cell microspheres are easier to achieve large-scale production of uniform size, and can be imaged and analyzed by optical, fluorescent, confocal microscopy ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/06G01N1/30G01N1/36
CPCG01N1/06G01N1/30G01N1/36G01N2001/302
Inventor 冯珊珊娄东晓魏孟超王恒李静谢泳娇王汝仙薛有安
Owner SUNSHINE LAKE PHARM CO LTD
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