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Double-fixation embedding sample preparation method after integral immunization

A double fixation and overall technology, applied in the field of transmission electron microscopy, can solve the problems of poor tissue structure, poor fixation, tissue structure and antigenic activity, etc., to improve the success rate and solve the effect of incompatibility

Pending Publication Date: 2021-09-21
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Since only paraformaldehyde is used as a fixative, immunolabeled samples are usually not fixed well, and the resulting tissue structure is poor, and even some antigens are very sensitive and easy to deform. The tissue structure and antigenic activity cannot be obtained in the whole experimental process. good preservation

Method used

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  • Double-fixation embedding sample preparation method after integral immunization
  • Double-fixation embedding sample preparation method after integral immunization
  • Double-fixation embedding sample preparation method after integral immunization

Examples

Experimental program
Comparison scheme
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Embodiment 1

[0036] Sample information: Radix japonicus root nodule

[0037] Double-fixed embedding sample preparation process after whole body immunization:

[0038] 1. The first fixation: the tissue is cut into small pieces, the maximum thickness of which is not more than 500 microns, placed in a phosphate buffer solution with a mass fraction of 4% paraformaldehyde and stored for 2 hours at 4°C;

[0039] 2. The first cleaning: wash the fixed material with phosphate buffer solution for 5 times, each time for 5 minutes, at 4°C;

[0040] 3. Digestion of cell walls: enzymatic hydrolysis solution prepared in phosphate buffer (containing a final concentration of 0.15% mass fraction Macerozyme, 2mM MES, pH5.0), put the tissue block in step 2 in the enzymatic hydrolysis solution, and digest the material at room temperature 45min, then washed with phosphate buffer solution at 4°C for 3 times for 5min each time;

[0041] 4. Membrane permeation: Place the material in step 3 in a phosphate buffer ...

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Abstract

The invention discloses a double-fixation embedding sample preparation method after integral immunization. Before conventional sample preparation of a transmission electron microscope sample, a first antibody and a second antibody are combined with an antigen, and then the transmission electron microscope sample is prepared. Before conventional sample preparation, the primary antibody and the secondary antibody are combined with the antigen, so that the activity problem of the antigen does not need to be worried, channels are opened on cell walls and cell membranes through macerozyme and Triton-100, the difficult problem that the antigen is in poor contact with the primary antibody and the secondary antibody is solved, and the marking success rate is increased. Glutaraldehyde and osmium tetroxide can be used in the following steps according to the sample preparation process of the biological tissue block, so that the problem that antigen activity preservation and tissue structure preservation are difficult to be compatible is solved.

Description

Technical field: [0001] The invention belongs to the field of transmission electron microscopy, and in particular relates to a double-fixed embedding sample preparation method after whole-body immunization. Background technique: [0002] Transmission electron microscopy is the highest resolution microscope available. The ultrastructure of the tissue can be observed using transmission electron microscopy, combined with enzyme localization, ion localization, and immune nano-gold labeling techniques, the expected distribution of enzymes, specific ions, and antigens (mostly proteins) in organelles can be known. [0003] Transmission electron microscopy requires samples to be very thin and free of water, so biological tissues usually need to go through the process of aldehyde fixation-osmium tetroxide fixation-dehydration-embedding-ultra-thin section-heavy metal staining before they can be observed in the transmission electron microscope. The conventional immunolabeling techniqu...

Claims

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Application Information

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IPC IPC(8): G01N1/28G01N1/36G01N1/30G01N23/04G01N23/20008
CPCG01N1/30G01N1/36G01N23/04G01N23/20008G01N2001/364
Inventor 邓汝芳刘旭陈雅平刘乐如徐信兰贾永霞胡晓颖
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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