Can I keep root/leaf samples in fridge (-80 C 0r -20 C) for several days? Or should I immediately prepare sample for Transmision Electronic Microscope (TEM) imaging?
Dear Narges,
I have never performed EM on plant tissue. I only work with mammal tissue and cell culture (mammal too). With these samples it is best to fixate cells immediately in 2% glutaraldehyde wit or without Formaldehyde. This can be left in fixative at 4°C for a long time. When it suits me best, i progress my samples to post fixation and embedding. Once again, for plant samples that might be different.
Hi Narges,
Do NOT freeze your plant samples (or any others) unless you are doing cryo-TEM.
Fix your root/leaf samples before storage. Leaves, depending on cuticle thickness, will need to be 'de-waxed' before/during fixation, roots will not need this.
In general, I recommend a variation of "Karnovsky's Fixative." 2%Glut, 4%ParaFormaldehyde in 0.1M cacodylate is the original recipe, I recommend using an iso-osmotic phosphate or citrate buffer since arsenic is toxic.
Many references for fixing and processing plant materials should be available, these will also have ways to deal with the leaf's cuticle.
The cell wall's chemical resistance can be overcome by adding 0.1% (v/v) dimethylsulfoxide (DMSO) to your fixative(s).
Lorenzo
Hi Narges!
I think that is very important to do not freeze the samples if you are studying the ultrastructure by TEM using conventional methods (ultrathin sections).
I also recommend you to do all the process of prefixing (Karnowsky´s fixative and embedding) in a system that allows you to work in a low vaccum conditions. These conditions help the penetration of the glutaraldehyde and the epoxy resin.
You also have to do a postfixing with 1% osmium tetroxyde for 24 hours in order to fix the membranes.
Because of the vegetal cell Wall you have to increase all the incubations times with alcohols, propylene oxid and epoxy resin.
All the procedure from prefixing to the 70% ethanol shoud be done at 4ºC.
I am sure that there are several publications about your question. You should do a review.
I hope these tips will help you
Good luck!
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