This method address the challenges in Deep Fluorescence Observation in Rice Shoots, such as a limited tissue penetration of the clearing solution and the real resolution under confocal microscope. The main advantage of this technique is a structure observation of much large tissues from a macro perspective, even prompts with hard and thick tissues, such as adult-erized shoots. This method applies to deep imaging of hard and thick tissues in wide plant species.
It can help accelerate the discovery of new phenomena in plant biological research. To begin, cut the roots carefully without damaging the plants and wash them with water to remove the dirt. Now, use tweezers to peel off the old outer leaves.
To avoid crushing the cells, use a single-edged razor with a sliding motion to cut the tissue from the shoot, apical meristem, or young panicle to the base. If the position of the shoot apical meristem or young panicle is not visible, cut it longer. The rice shoot has an oval cross section.
Use a double edged razor to shave off one side of the oval thinly, in the direction of its long axis. Confirm the position of the shoot apical meristem or young panicle by the appearance of a white-ish color in the sample and trim off the excess tissue. Put the sample in a 1.5 milliliter micro centrifuge tube containing one milliliter of fixative solution.
Cut a 2.5 square centimeter piece of paraffin film and shape it into a ball. Now, place these balls on top of the samples to prevent them from floating out of the fixative solution. Place the tube containing the sample in a desiccator.
Close the lid of the desiccator and start the vacuum pump to slowly depressurize the inside of the desiccator until minus 0.095 megapascals pressure is reached. After closing the desiccator at minus 0.095 megapascals, turn off the vacuum pump and allow it to stand for 30 minutes at room temperature. Open the desiccator slightly and slowly return it to atmospheric pressure.
If the sample is correctly fixed it sinks into the fixative solution. And if it does not sink, reduce the pressure again. Remove the paraffin film.
Close the lid, and keep the tubes overnight at four degrees Celsius. Using lint-free wipes, remove the excess fixative solution from the sample and fix the sample on a vibrating micro-slicer tray with instant glue. Place the sample on a tray with the flat side down which was shaved off during sampling and set the trimming conditions according to the fixed sample.
Adjust the sample position with the reverse or forward button and the up or down button and fill the tray with deionized water until all the samples are immersed. After filling the tray, press the start button and place the trimmed samples on a glass slide with a drop of PBS. Check the sample containing the target site under a stereo microscope and transfer the trimmed sample to a multi-well plate with one milliliter of PBS.
Remove the PBS from the multi-well plate. Add fresh PBS and let it stand for one minute. Remove PBS and add the CS.Place the multi-well plate containing the sample in a desiccator and close the lid.
Then start the vacuum pump to depressurize the inside of the desiccator until minus 0.09 megapascals, slowly. After closing the desiccator at minus 0.09 megapascals, turn off the vacuum pump and let it stand for one hour at room temperature. Open the desiccator slightly and slowly return it to atmospheric pressure.
Pour deionized water into the gap between the wells to prevent the evaporation of the CS and store the multi-well plates at room temperature in the dark for clearing. When the CS turns green, replace it with a fresh solution. The un-trimmed samples remained green and did not become transparent after three months of treatment with the CS.In samples that were peeled off all leaves the inter nodes turned white after one week, although the nodes remained green.
The sample did not become transparent after four weeks. The hand trimmed samples became white after one week of CS treatment but did not become transparent after four weeks. The samples trimmed to 130 micrometers thickness became translucent after one week of CS treatment.
The sample was almost transparent after two weeks. In the node, the observable depth after one week of the CS treatment was minus 60 micrometers and minus 90 micrometers after two weeks. Fluorescent signals were observed at a depth of minus 80 micrometers at three weeks and at a depth of minus 100 micrometers at four weeks.
In the internode, fluorescent signals were observed at a depth of minus 60 micrometers without the CS treatment. The observable depth was minus 90 micrometers after one week to three weeks of the CS treatment and minus 100 micrometers after four weeks. In the leaves fluorescent signals were observed at a depth of minus 90 micrometers without CS treatment, but the brightness was weaker than other tissues.
After one week, the signals were brighter and observed at a depth of minus 120 micrometers. The expression of the transcription factor OS MASU 15 mOrange was observed in young Panicle, leaf, node, internode, and fleurette. The key is to find the best conditions for the plants or tissues, for instance, period and vacuum pressure, or position, and CS treatment, timing, thickness, speed and appropriate staining solution.
The combination of timing and curing technology allows spatial temporal observation of gene expression pattern and intercellular communication in multiple tissues of a single section.
