Ele Mira

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• Answer added in Microscopy
  12 Vegetal Tissue preparation for TEM
  By Ele Mira · Université des Antilles et de la Guyane
  Ele Mira · Université des Antilles et de la Guyane

  Hi First, thanks for your reply So, Mr Wolfgang Muss, i can tell you i made some research on google and specific litterature before sending this p... [more]

  Hi First, thanks for your reply So, Mr Wolfgang Muss, i can tell you i made some research on google and specific litterature before sending this post, and try a lot of "recipies"but none of them gave perferct results. In my lab, microscopists are only physicians or animalist so they can't help me with vegetal stuffs and the specialized labs provides a lot of expensive formation but keep their technical advices... I agree that my post lack of precision an will fastly correct that. First, I work on lamina of the leaves of angiosperm woody species, i consider 20 different species of tropical dry, wet and coastal forest.In their leaves, i cut samples of 1mmx3mm, avoiding midrib The best result obtained was following this protocol. I do fixation in a glutaraldehyde 4% solution during 2h (at ambiant T° because cells of leaves of tropical species are known to collapse at 6°) and change with a NaH2PO4 buffer at 25mM (3 baths of 20 min) as my field stations are far from my lab, i directlly do the fixation on the field, tho avoid any degradation in my tissus i do deshydratation with successive bath of absolute ethanol (gradation of 10°, 30° 50° 70° 90° 100 100 100), but not under vacuum i make a 1/2h bath of pure propylenoxyde and then, i begin the infiltration using PO and a fresh mix of resin (as I said, Agar resin kit, low viscosity) 1bath 24h 2PO:1resin; under agitation 24h 1PO:1resin under agitation 24h 1PO:2resin under agitation 24h x3 pure resin under vacuum in every bath, I put 10x the volume of my sample Polymerisation is made at 70°during 12h ialready try classical spurr resin , the impregnation was very bad so i thought it was because of the high viscosity of this product I also already try short times of impregnation but the problem of penetration is worst I really care about the flotabillity of my samples during all the operations, they never float when i change baths. As i told you, that's particular structure wich are always bad embedded: cells with high cellular walls, glandular cells. Maybe the using of T-X100 as suggested by Euan James could help to improve the permeability of theses structure. It's hard to do the fixation under vacuum because some of my plants grow far away from my lab and the transport dry them (and i guess that proteic digestion begin few minutes after the cut) so in order to preservate the integrity of my tissues, i make the fixation directlly in the field. I'm the first to integrate microscopic approch in my lab so my financial means are very limited (i'm a little PhD student so my boss let me play with little cheap resins but that's all), of course Freezing/cryosectioning or Freeze substitution sounds good but we doesn't have the equipment to consider them seriouslly and i'm in a little island far from everything, the transport of fresh leaves to an equiped lab is impossible so i make what i can with what i have. So thank you very much for all your messages, that was my first post on this site and i'm surprised to receive so much answers.
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• Question asked in Microscopy
  12 Vegetal Tissue preparation for TEM
  I have some problems in the embedding process in foliar tissue of tropical species, and particularly with species containing glands and trichoms. Aft... [more]
  I have some problems in the embedding process in foliar tissue of tropical species, and particularly with species containing glands and trichoms. After fixing with glutaraldehyde, deshydratation and bath in epoxypropylene, I use the low viscosity resin kit from agar and despite that, the infiltration in the samples is pretty bad. I guess some molecular exchanges occurs during the impregnation and I would like to know if it's possible to avoid them. Has anyone some experience in vegetal tissue preparation ?
  By Ele Mira · Université des Antilles et de la Guyane
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