Are there any tips for obtaining clear images (20x) of cell files in each zone of Arabidopsis roots?
I normally fix them first in 96% ethanol and mount them in chloral hydrate. However, due to the ethanol, the roots are wrinkled, starting at the elongation zone (the meristem is okay).
Reducing the timing of ethanol clearing does not alter this effect, nor does using a lower percentage of ethanol, as observed in my samples with GUS-staining which are conserved at 70%.
I could try increasing the percentage, but this will be time consuming to apply to each sample.
Does anyone have any other ideas?
Look up Talbot and White Plant Methods 2013, 9:36
They compare several methods and even use Arabidopsis thaliana as one of their study species.
If you have access to a nice confocal microscope, all you need to do is stain the membranes (FM4-64) or use propidium iodide. No need to fix the tissue.
Skip the ethanol and directly mount in chloral hydrate for roots that are not stained with GUS. Else do not use higher than 70% ethanol. If your light microscope has good DIC optics/filter, you should be able to get very nice images, also from adult roots.
To avoid the use of chloral hydrate, which is a controlled substance in many countries, you can try the protocol described here: http://www.ncbi.nlm.nih.gov/pubmed/9006065
"For observation of whole mounts, both stained and unstained
roots were transferred to small Petri dishes containing 0.24 N HCl
in 20% methanol and incubated on a 57°C heat block for 15
minutes. This solution was replaced with 7% NaOH, 7% hydroxy-
lamine-HCl in 60% ethanol for 15 minutes at room temperature. (The hydroxylamine can be omitted with no decrease in effective
clearing). Roots were then rehydrated for 5 minutes each in 40%,
20% and 10% ethanol, and infiltrated for 15 minutes in 5% ethanol,
25% glycerol. Roots were mounted in 50% glycerol on glass micro-
scope slides."
I agree with Senthil, I have obtained beautiful images staining with propidium iodide and imaging with a confocal microscope.
Try Gustavo's protocol and use DIC to take the pictures.You can remove hyroxyl-amine. My lab use this protocol and DIC, and we get beautiful images.
Hi, Jolien, you can fix roots in 100% methanol (60°C) for 15 minutes, and thereafter VERY gradually add water till methanol concentration became 20%. Thereafter you must wash seedlings 2 times in pure water and incubate 5-15 minutes in chloral hydrate. Thereafter transfer roots to 50% glycerol and mount in glycerol. you will have a very nice picture! Please, add water very gradually!!!
This is a key step!
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