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Immunofluorescence method for rapidly detecting nucleolus protein positioning under the stress of heavy metal

A detection method, immunofluorescence technology, applied in the field of cell biology

Inactive Publication Date: 2010-02-17
TIANJIN NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of immunofluorescence techniques to study the effects of heavy metal stress on the function and dynamic changes of B23 protein during the nucleolar cycle has not been reported in the literature

Method used

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  • Immunofluorescence method for rapidly detecting nucleolus protein positioning under the stress of heavy metal
  • Immunofluorescence method for rapidly detecting nucleolus protein positioning under the stress of heavy metal

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Material culture takes broad bean as an example: when the main (side) root of broad bean is about 1.5cm long, use 10μM, 50μM, 100μM Cd 2+ Treatment 24h, 48h, 72h.

[0074] (1) Cut out the root tip meristem cells of Vicia faba plant after heavy metal treatment, fix in 4% paraformaldehyde for 2 hours; wash with PBS buffer solution;

[0075] (2) Use 2.5% cellulase + 2.5% pectinase enzyme solution to enzymolyze in a 37°C incubator; wash with PBS buffer and press into tablets;

[0076] (3) Transfer the root tip of the faba bean plant to a centrifuge tube, add PBS buffer solution dropwise, shake it by hand until most cells are free, use a dropper to absorb about 0.1ml of sample solution, and spread it evenly on a glass slide to disperse into single cells. After marking, let it air dry naturally for later use;

[0077] (4) soak the glass slide obtained in (3) in 1% TritonX-100 for 15 minutes; wash with PBS buffer;

[0078] (5) Drop 15 μl of the prepared primary antibody on ...

Embodiment 2

[0084] Material cultivation Taking garlic as an example, when the adventitious root of garlic is about 1.5cm long, use 10μM, 50μM, 100μM Pb 2+ Treatment 24h, 48h, 72h. Pb

[0085] Experimental steps:

[0086] (1) Cut out about 2 mm of the root tip after the heavy metal treatment, and fix it in 4% paraformaldehyde for 2 hours.

[0087] (2) Wash in PBS buffer (pH 7.0) 3 times (10 minutes each time).

[0088] (3) 2.5% cellulase + 2.5% pectinase, 37 ° C incubator enzymatic hydrolysis, microscopic inspection at any time during the enzymatic hydrolysis, until a large number of spherical protoplasts are produced to end the enzymatic hydrolysis.

[0089] (4) Wash with PBS buffer 3 times (10 minutes each time).

[0090] (5) Transfer the root tip to a 0.5ml centrifuge tube, add a small amount of PBS buffer dropwise (depending on the size of the sample), cover the centrifuge tube, shake it by hand until most cells are free. Use a dropper to draw about 0.2ml of sample solution, apply...

Embodiment 3

[0102] Changes of Nucleolar B23 Protein in Onion Root Tip Cells

[0103] Material culture takes onion as an example: when the adventitious root of onion is about 1.5cm long, use 10μM, 50μM, 100μM Al 3+ Treatment 24h, 48h, 72h.

[0104] Experimental steps:

[0105] (1) Cut out about 2 mm of the root tip after the heavy metal treatment, and fix it in 4% paraformaldehyde for 2 hours.

[0106] (2) Wash in PBS buffer (pH 7.0) 3 times (15 minutes each time).

[0107] (3) 2.5% cellulase + 2.5% pectinase, 37 ° C incubator enzymatic hydrolysis, microscopic inspection at any time during the enzymatic hydrolysis, until a large number of spherical protoplasts are produced to end the enzymatic hydrolysis.

[0108] (4) Wash with PBS buffer 3 times (15 minutes each time).

[0109] (5) Transfer the root tip to a 0.5ml centrifuge tube, add a small amount of PBS buffer dropwise (depending on the size of the sample), cover the centrifuge tube, shake it by hand until most cells are free. Use...

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Abstract

The invention discloses an immunofluorescence method for detecting plant cell nucleolus B23 protein positioning under the stress of heavy metal, which comprises the following steps: cutting a plant root meristematic tissue cell processed by heavy metal and fixing for 1 hour in 4% paraformaldehyde; carrying out enzyme solution enzymolysis, using a burette to sucke out 0.1ml of sample liquid and uniformly spreading the sample liquid on a glass slide to be dispersed into a single cell; marking and then naturally drying by air; soaking in1 % TritonX-100 for 15 minutes; then dropwise adding 15 microliters of prepared first antibodies, covering a cover glass slide and hatching for 1 hour in a thermostat at 37 DEG C; dropwise dropping 15 microliters of prepared second antibodies; processing and then dropwise dropping 15 microliters of DAPI; finally dropwise adding 5 microliters of quenching preventing agent on a glass slide material, covering the cover glass slide and sealing the periphery ofthe cover glass slide with nail polish; and observing under a fluorescence microscope after 1 hour. The method has the characteristics of high specificity, accurate experimental result, and the likeand greatly improves the quantity of observation cells. The method provides a precise detecting method for discussing a heavy metal toxic cell mechanism and has wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of cell biology, and relates to a localization method for rapidly detecting nucleolar protein changes under heavy metal stress. More specifically, it is an immunofluorescence method to detect the nucleolus B23 protein localization in plant cells under heavy metal stress. This method provides new technical support for the study of the mechanism of heavy metal poisoning of plant cells. technical background [0002] In recent years, with the rapid development of modern industry, mining and ore smelting, unreasonable application of chemical fertilizers and pesticides, discharge of "three wastes" and urban domestic waste, and agricultural use of sludge have all led to increasingly serious environmental pollution problems, especially Pollutants containing heavy metals (As, Cd, Co, Cr, Cu, Hg, Mn, Ni, Pb, Zn, etc.) enter the environment through various channels, and endanger the health of animals and humans throug...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64G01N33/68G01N33/577
Inventor 刘东华秦蓉张闪闪张慧敏
Owner TIANJIN NORMAL UNIVERSITY
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