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
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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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