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Immunofluorescence method for detecting nucleolus fibrin positioning under heavy metal stress

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

Inactive Publication Date: 2012-11-07
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 nucleolar fibrils has not been reported in the literature

Method used

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  • Immunofluorescence method for detecting nucleolus fibrin positioning under heavy metal stress
  • Immunofluorescence method for detecting nucleolus fibrin positioning under heavy metal stress

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Material culture takes broad bean as an example: when the main (lateral) root of broad bean is about 1.5cm long, use 10μM, 50μM, 100μM Cd 2+ Treatment 24h, 48h, 72h.

[0078] (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;

[0079] (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;

[0080] (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;

[0081] (4) soak the glass slide obtained in (3) in 1% TritonX-100 for 15 minutes; wash with PBS buffer;

[0082] (5) Drop 15 μl of the prepared primary antibody ...

Embodiment 2

[0088] For material cultivation, take sunflower as an example. When the root length of sunflower is about 1.5cm, use 10μM, 50μM, 100μM Pb 2+ Treatment 24h, 48h, 72h.

[0089] Experimental steps:

[0090] (1) Cut out about 2 mm of the root tip after the heavy metal treatment, and fix it in 4% paraformaldehyde for 2 hours.

[0091] (2) Wash 3 times in PBS buffer (pH 7.0) (10 minutes each time).

[0092] (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.

[0093] (4) Wash with PBS buffer 3 times (10 minutes each time).

[0094] (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 it ev...

Embodiment 3

[0106] Changes of Nucleolar Fibrillarin Protein in Onion Root Tip Cells

[0107] 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.

[0108] Experimental steps:

[0109] (l) Cut out about 2 mm of the root tip after the heavy metal treatment, and fix it in 4% paraformaldehyde for 2 hours.

[0110] (2) Wash 3 times in PBS buffer (pH 7.0) (15 minutes each time).

[0111] (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.

[0112] (4) Wash with PBS buffer 3 times (15 minutes each time).

[0113] (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 fr...

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Abstract

The invention discloses an immunofluorescence method for detecting plant cell nucleolus protein positioning under heavy metal stress. The method comprises the following steps: cutting plant root tip meristem cells after heavy metal treatment; fixing the cells in 4 percent paraformaldehyde for 1 hour; adopting enzyme fluid to perform enzymolysis; sucking about 0.1 millimeter of sample solution with a dropper; uniformly smearing the sample solution on a glass slide to disperse the sample solution into single cells; naturally airing the obtained product after labeling; soaking the obtained product in 1 percent TritonX-100 for 15 minutes; dropping 15 mu l of a first antibody prepared; covering the obtained product with a cover slide; incubating the obtained product for 1 hour in a warm box at37 DEG C; dropping 15 mu l of a second antibody prepared; dropping 15 mu l of DAPI after treatment; dropping 5 mu l of anti-quenching agent on a glass slide material; covering the obtained product with the cover slide; sealing the periphery of the cover slide with nail polish; and observing the obtained product under a fluorescence microscope after 1 hour. The detection method has the characteristics of high specificity, accurate experimental result, great increase in the number of observation cells, and the like. The method provides a precise detection method for studying the cell poisoning mechanism of heavy metal, and the detection method has broad application prospects.

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 nucleolar fibril 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] With the massive development of mineral resources, the widespread use of pesticides and chemical fertilizers, and the agricultural use of urban sludge and sewage, the pollution of heavy metals to soil and water is becoming more and more serious. Excessive heavy metals (Cd, Cr, Hg, Pb, Cu, Zn, Ag, Sn, etc.) enter the environment, participate in the cycle of water body-soil-biological system, accumulate in plants through the absorption of plants, and endanger the health of animals and humans through t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/68G01N21/64G01N33/577G01N1/28
Inventor 刘东华张闪闪张慧敏秦蓉
Owner TIANJIN NORMAL UNIVERSITY