Semithin sections are usually a prerequisite for TEM-examination of ultrathin sections. They are prepared in advance and in most EM-Labs only stained by Toluidine blue because only an approximate morphological information on the area to be sectioned for ultrathins is sufficient. I am working on a publication project on "Semithin [sometimes also referred to as "thin"] Resin Section Stainings" usable also for diagnostic purposes ("diagnostic staining"). I would like to ask interested colleagues whether they would like to share their
i) preferred stainings,
ii) indicate the resin used,
iii) indicate whether staining should be performed only for orientation or also as a "diagnostic tool".
Your (even confidential) reply would help me a lot in defining whether there is a demand of such staining method or not as well as - naturally only as an approximation - how many (T)EM-Labs (all over the world or at least a representative part of it would be great) are using other stains than Toluidine blue.
Thank you in advance.
Dear Gregory, thank you for the first / your input.
Just adding the following consideration:
certainly experienced observers are satisfied with only a TB-(Toluidine Blue) stain (usually 1% TB in alkalinized [+ e.g. 2% Borax] dist. water, applied to sections on object slide under heat). TB-staining procedure that way (either obj.slide on hot-plate or heating the obj. slide with the aid of a "flame", e.g. Bunsen burner, slide warmer, microtorch,etc.) is really easy and fast...but only hardly or subtly can be "standardized". The effects after TB-stain on resin("plastic") sections -if any - are questionable and not always (if not for the most part) achievable.
I intend to refer to this ResearchGate-Thread in the planned publication, so everybody interested is i) invited cordially as well as ii) is/will be acknowledged adequately - hopefully-. After some replies more I plan to provide a sample of evidence for the conception of presenting a "diagnostic" semithin resin section stain. Thanking in advance for your interest, best regards, Wolfgang
Dear Hans, thank you,
allow for three short questions:
i) your resin (if Epon, please specify source/vendor)
ii) did you get - with your staining procedure (assuming this to be the classical "Richardson stain"- at any time "metachromatic staining" of structures (means: blue, red, violet, for instance)...and:
iii) have you tried anytime an AMbF (= Azure II - Methylenblue - basic Fuchsin), (there exist a wide range of "similar" staining sequences, sometimes also decribed as bFMA, MB-AA-bF, and so on...)
Thanks for your time and collegiality,
best regards, Wolfgang
We are working on research regarding epidermal blisters. Skin biopts are embedded in EPON (5/5) or freeze substituted to HM20- resin.
Semithin sections (500 nm) on poly-L Lysine coated coverslips are stained at 80°C for 15 (EPON) to 45 (HM20) seconds. Rinse w/ tap-water and demiwater.
I do not use them in a classical diagnostic way but, only to see if I'm cutting the blister region. However, our pathologist can use these TB-stainings in a diagnostic way. E.g for blister typing or finding a basal cell carcinoma in the dermis.
The staining is used for orientation as well.
Good morning, dear Hannes, thank you very much!
Unfortunately the original title of my question
["Semithin Sections not only as prerequisite for (T)EM: use for diagnostic purposes too? (staining methods survey)" ] was "rearranged" by "an editor", so it does not reflect any more the intended purpose. Nevertheless hoping that at least some researchers and colleagues in the field will have some answers more on this topic.
HAve a nice weekend, best regards, Wolfgang
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