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, it is said to be pH4.5, then in the recipe title, it is again 4.5 but in the description you say bring it to 6.8. Then figure 4, you say the peels are floating in HEPES buffer pH6.8. Moreover in the associated Nature plants paper, you say make a HEPES buffer solution pH7 + Tween-20 0.1%, which is not mentionned in this present protocol.
I am especially asking this question because my first trials went quite well during the peeling stage, but when layed in the buffer, the peels would inevitably curl up into little tubes really hard to lay flat on a microscopy slide and the fact that this behaviour is not mentionned in your protocol leads me to think that you have not encountered such problem. I am thus suspecting my buffer, which does not contain the Tween-20 and is at pH6.8.
In brief can you clear out the HEPES and sodium acetate buffer recipes please?
Cheers!
William Nicolas
1/24/2018 9:48:51 AM Reply