Dear authors, I am currently trying to produce onion epidermal...

protocol Protocol: Biomechanical Characterization of Onion Epidermal Cell Walls
Dear authors, I am currently trying to produce onion epidermal cell wall peels, exactly like in your Nature plants paper. This brought me to your detailed Bio-protocol paper. First of all, The pH at which the HEPES solution must be is not clear at all. In the materials in reagents section Read more Read less more less
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William Jules Nicolas Answered Jan 25, 2018

HHMI - UCLA LA

Ok thanks a lot, putting them with the cuticle facing up fixed the curling problem. Staining with DAPI also seems to indeed show the absence of the nuclei in the peeled part. The nuclei nonetheless remain in the thicker bordering parts, which is good. Thanks for the advice ! William Nicolas
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Daniel J. Cosgrove Author Answered Jan 25, 2018

Department of Biology and Center for LignoCellulose Structure and Formation, 208 Mueller Laboratory, Pennsylvania State University, USA, USA,

Thanks for your query and we are happy to clarify. In the Materials and Recipes sections, the pH of the HEPES and acetate buffers were incorrectly amended during the last stage of bio-protocol publication, and we missed this editorial change. HEPES buffer should be pH 6.8 and acetate buffer should be pH 4.5, throughout. Sorry for the confusion and we will ask bio-protocol to correct these typos. About curling: indeed the peeled strips have a strong tendency to curl. As described in step A.8 and shown in Figure 4B, it is important to leave chunks of subepidermal tissue at the two ends of the peel (like barbells). This helps a lot with the handling. Also, when you float the peels on a solution, ALWAYS place them with cuticle side up (facing the air). If you place the cuticle side down, they will curl. The power of surface tension of water! With a fine pair of forceps they can be uncurled under a dissecting scope, but it is challenging. The issue with curling should have been made clearer in the protocol. The curling behavior is not likely related to the solution composition. Also, we note that the tendency to curl varies greatly among onions, so try more than one onion. Good luck.
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