We want to characterise the ultrastructure of some glands in Lepidoptera using TEM (we would like to observe the cell types, tubules, etc), but our insects are seasonal, with only one generation per year. Is it possible to store them (frozen, perhaps) and fix them some months later? Or must they be alive? Do cells get deformed?
Thank you in advance.
Dear Ricardo,
as far as I know, if you want to have beautiful results samples must be alive when you make the dissection and put them into the fixative. Freezing will damage your samples.
Sincerlely
Francesco
Dear Ricardo,
I agree with the answer given to you from Francesco.
I may also suggest you to cut your specimen in small pieces, if you can, to improve the fixation, dehydration and the resin infiltration processes.
Sincerely,
Veronica
Thank you for your answers. I guess we would need to find a technician to process our insects as soon as possible.
I agree with both answers above. But I can add that after fixing live disssected specimens in glutaraldehyde you may store them in this fixative for a few weeks before processing them further for TEM.
Just to add few words to the previous answer. If you plan to store your sampIes in fixative, I would not do it in glutaraldehyde. It makes them too hard and brittle. You can fix them in glutar and store them in 4% formaldehyde in 0.1-0.2 M Hepes for years.
You would do well to consult the best images of insects in the literature (Microscopic Anatomy of Invertebrates, ed. Harrison) and not use standard "mouse" fixatives, but rather mixtures of formaldehyde, glutaraldehyde and even small amounts of acrolein. The disassembling and dissection of the animal should happen within the fixative to avoid any effects of air. Keep the tissue cubes as small as compatible with needed orientation.
Dear Ricardo,
as the previously posted replies contain much information about what to do and what shouldn't be done because of introducing artifacts one (we) should perhaps also know which stage of the Lepidoptera you want to examine. Since in some places of the globe are also you naturally could "put into and dissect in fixative" the "samples" which will result in a certain stress for the insects... (not knowing which you want to examine). So IMHO, if you are going to treat your "samples" the latter way you might induce "physiological and/or metabolic" artifacts due to stress of the animals during (hampered or delayed) fixation. To get rid of such you might (think over or) intend to initially anesthesize the butterflies or moths (cf. John E.COOPER, ILAR Journal, 2011; @ http://ilarjournal.oxfordjournals.org/content/52/2/196.full.pdf), which I have attached here for your convenience.
You'll find there some interesting viewpoints (e. g. adverse effects of ether, GP/GET [Good Practice / Good Experimental Techniques], "What is out, What is in", and last but not least, about "invertebrate welfare").
There also could be added specific recipes on fixatives etc. but general info on this has been given already in the previous posts.
Best of luck and regards.
Your answers are quite valuable. It's a relief that I could store the specimens at least for a few weeks, while we get someone to do the rest.
I have been reading about the different fixatives, and it has been a little overwhelming, with all the variants, etc. I will consider solutions with HEPES and formaldehyde (We haven't got acrolein).
Dr Wolfgang, thank you very much for the article, I'm reading it, and it is really interesting. I had not considered throwing the insects in the fixative without their being anaesthetised, not only because of the possible artefacts, but also because I too consider that inhumane.
Dear Ricardo,
you're welcome. It is always good to see that GP/GET/GLP/ are not only words.... (saying this as an
Thank you. According to what I have been reading here and on some articles, yes, formaldehyde and glutaraldehyde will be the main components, and we will store our samples in HEPES. I sill have some doubts, because I've read that phosphate-buffered fixatives sometimes form precipitates that may interfere with the observation... what has been your experience in this respect?
Best regards
I'm not sure if this would work for you, but I used to be a tech in a lab where we kept our insects (or their harvested tissues) in some % of glycerol (cryoprotectant) until we were ready to process them for TEM. We were interested in insect flight muscles. Sorry I can't remember much else.
Good luck!
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