Gumnosperm pollen tube microfilament framework fluorescent marking method and its uses
A gymnosperm and fluorescent labeling technology, applied in plant cells and other directions, can solve the problem of lag in biological research, and achieve the effect of good labeling effect, simple operation and reduced damage.
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Embodiment 1
[0025] Example 1. Fluorescent labeling of the microfilament skeleton of white rod pollen tubes and its microscopic observation
[0026] Firstly, the method of the present invention is used to carry out fluorescent labeling to the microfilament skeleton of the white pole pollen tube, comprising the following steps:
[0027] 1) Collect the white stem pollen tube samples to be marked, and prepare them in freshly prepared 50mM Pipes buffer (50mM PIPES, 0.5mM MgCl) containing 3% paraformaldehyde 2 , 1mM EGTA, pH 6.9) and quickly pumped and fixed for 30min;
[0028] 2) Wash 3 times with 50mM Pipes buffer to remove residual paraformaldehyde;
[0029] 3) Put the pollen tubes in 0.25% glycerol and infiltrate at room temperature for 40 minutes;
[0030] 4) wash 3 times with 50mM Pipes buffer;
[0031] 5) Incubate the pollen tube with 200 nM TRITC-phalloidin (Sigma, USA) in the dark at room temperature for 1 h. Then, drop into 50% glycerol containing 0.2% Propyl gallate (Sigma Compan...
Embodiment 2
[0032] Example 2. Fluorescence labeling and microscope observation of the microfilament skeleton of Pine pine pollen tube
[0033] Firstly, the method of the present invention carries out fluorescent labeling to the pine pollen tube microfilament skeleton, comprising the following steps:
[0034] 1) Collect the white bark pine pollen tube samples to be marked, and prepare them in freshly prepared 60mM Pipes buffer (60mM PIPES, 0.5mM MgCl) containing 4% paraformaldehyde 2 , 1mM EGTA, pH 6.9) in fast pumping and fixation for 50min;
[0035] 2) Washing 4 times with 60mM Pipes buffer to remove residual paraformaldehyde;
[0036] 3) Put the pollen tubes in 0.2% glycerin and infiltrate at room temperature for 60 minutes;
[0037] 4) wash 4 times with 60mM Pipes buffer;
[0038] 5) Incubate the pollen tubes with 250 nM TRITC-phalloidin in the dark at room temperature for 1.5 h. Then, drop in 60% glycerin containing 0.2% Propyl gallate to the fluorescently labeled pollen tube samp...
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