I'm trying to determine the grade of lignification in the peduncles of different angiosperms using ImageJ. First I embedded the stems in paraffin and stained them with Etzold's solution (beautiful staining) and phloroglucinol. However the parenchyma was never intact. Now, I embedded them in LR-white and the stems are mostly intact. However, I can not use ethanol based stains (phloroglucinol) and astra blue (used in Etzold's ) is not strong enough to penetrate. I found a protocol for phloroglucinol where they dissolve it directly in HCl so that problem is hopefully solved. But, I am still looking for a staining that is longlasting and distinctly highlights the lignin. Can anyone think of a staining?
First off when sectioning plant tissue, fixation, infiltration time, and section thickness is all important. For paraffin we routinely section citrus stems at 10-12microM. If you're just staining them the you could also consider Spurr's resin, but you would not be able to do any efficient antibody staining, should you want to head that direction without significantly etching first. There are commercially available antibodies to lignin so you could design an assay that uses fluorescence, but you need to be aware of any autofluorescence and chose contesting fluorophores. Antibody labeling is more specific and you could even conjugate them to enzymes and develop it with a substrate if you prefer a more kilometric route over fluorescence.
Thank you so much for your answer. I also want to measure the thickness of the lignified cell walls and count the amount of cells with lignin. So I section them als thin as possible (currently 4 um). I'm gonna look into the Spurr's resin and the antibodies. Again, thank you for your suggestions!
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