Immunofluorescence staining method for paraffin embedded three-dimensional suspension cell clusters

A technology of immunofluorescence staining and suspension cells, which is applied in the field of immunofluorescence staining of paraffin-embedded three-dimensional suspension cell clusters. Good staining effect, saving reagents and antibodies

Pending Publication Date: 2020-05-29
DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the immunofluorescent staining methods for 3D suspension cell clusters (spheres) mainly include: (1) centrifugation; the disadvantage is that centrifugation will cause some loss of cell clusters, damage to cells, loose deformation, and insufficient staining of cells inside the cell clusters.
(2) Staining method with the help of a staining device; the disadvantage is that the removal of residual liquid is not complete or the cell mass is easy to dry out. One device can only do one set of sampl

Method used

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  • Immunofluorescence staining method for paraffin embedded three-dimensional suspension cell clusters
  • Immunofluorescence staining method for paraffin embedded three-dimensional suspension cell clusters
  • Immunofluorescence staining method for paraffin embedded three-dimensional suspension cell clusters

Examples

Experimental program
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Effect test

Embodiment 1

[0036] Example 1: Paraffin-embedded 3D cell cluster immunofluorescence staining

[0037] 1. Experimental materials

[0038] Cell: Human embryonic stem cell H9

[0039] Reagents: 75% alcohol, cell culture medium, Matrigel, PBS, 4% formaldehyde fixative, Triton X-100, absolute ethanol, xylene, EDTA, PBS, Goat Serum, antibody, dH 2 O, DAPI

[0040] Equipment: cell incubator (Thermo 150i), ultra-clean workbench (Suzhou Antai), Beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), inverted fluorescence microscope (LeicaDM i8) ), orbital shaker (Chemglass), 4 degree refrigerator (Haier HYC390), negative 20 degree refrigerator (Haier), negative pressure suction device (diving), pipette, microwave oven, paraffin embedding machine, paraffin slicer, automatic Dewaxing and rehydration instrument, ice maker

[0041] 2. Experimental method

[0042] 1. Cell cluster sample preparation

[0043] The cells were passaged into a low-adsorption 6-well plate (Corning, 3...

Embodiment 2

[0077] Example 2: Paraffin-embedded 3D cell cluster immunofluorescence staining

[0078] 1. Experimental materials

[0079] Cells: Human embryonic stem cell H9 differentiated pancreatic islet cell cluster

[0080] Reagents: 75% alcohol, cell culture medium, Matrigel, PBS, 4% formaldehyde fixative, Triton X-100, absolute ethanol, xylene, EDTA, PBS, Goat Serum, antibody, dH2O, DAPI

[0081] Equipment: cell incubator (Thermo 150i), ultra-clean workbench (Suzhou Antai), Beckman centrifuge (X-15R), cell counter (Countstar), inverted microscope (Leica DM IL LED), inverted fluorescence microscope (LeicaDM i8) ), orbital shaker (Chemglass), 4 degree refrigerator (Haier HYC390), negative 20 degree refrigerator (Haier), negative pressure suction device (diving), pipette gun (eppendorf), microwave oven, paraffin embedding machine, paraffin section Machine, automatic dewaxing and rehydration instrument, ice maker

[0082] 2. Experimental method

[0083] The specific experimental method is the same...

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Abstract

The invention relates to an immunofluorescence staining method of paraffin embedded three-dimensional suspension cell clusters. The method comprises the following steps: pretreating the three-dimensional suspension cell clusters, immersing into paraffin liquid to embed sections, carrying out antigen repair, and carrying out immunofluorescence staining. According to the method provided by the invention, cells in the cell cluster are fully dyed, the result is accurate, the overall protein expression and distribution condition of the suspension cell cluster can be observed, and sample observationcan be carried out only by a common fluorescence microscope.

Description

Technical field [0001] The invention relates to the field of cell biology and related detection, in particular to an immunofluorescence staining method for paraffin-embedded three-dimensional suspended cell clusters. Background technique [0002] Immunofluorescence technique (Immunofluorescence technique) is one of the earliest developed labeling immunological techniques. It is a technology established on the basis of immunology, biochemistry and microscopy technology. This method combines antibody molecules with some tracer substances, and uses antigen-antibody reactions to locate antigenic substances in tissues or cells. [0003] At present, the immunofluorescence staining methods for 3D suspension cell clusters (spheres) mainly include: (1) Centrifugation; the defect is that centrifugation will cause partial cell cluster loss, cell damage, disintegration and deformation, and insufficient staining of cells inside the cell cluster. (2) Staining method with the aid of a staining d...

Claims

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Application Information

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IPC IPC(8): G01N1/30G01N33/533
CPCG01N1/30G01N33/533
Inventor 宋益哲曹易照欧镇生粱德灿李景秋刘靖凌伊王慧陈小锋李文佳
Owner DONGGUAN HEC BIOPHARMACEUTICAL R&D CO LTD
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