Hi,
Does anybody have any experience with histologically processing arterial plaque tissue which may contain calcified regions?
All advice is welcome!
Robbie
Thank you very much for your response!
Would you have a protocol/reference for decalcification of arteries or perhaps a suggested concentration? Finally, will formerly calcified regions be clearly identified after the decalcification process? Thank you again.
Any further advice from people is also welcome.
Robert, it may be possible to use - besides the decalcification solutions already mentioned by Elizabeth E LeClair - also HNO3, formic acid, citric acid and last but not least (as used also for decalcification of electric kettles), amido-sulfonic acid. Every chemical has its Pro's and con's and must be applied properly to (better) fixed experimental tissue (otherwise you might find severe deterioration of tissue components).
I remember to have two or three times used ('colloidal') Lanthanum (La(NO3)3·6 H2O or La(NO3)3 solution*) after fixation [buffered FA-GA] of calcified tissues (e. g., mineralized hyaline cartilage and also some blood vessel preparations (done for LM & consecutive correlative electron microscopy). The effect consisted of exchange / substitution of calcium (ions/atoms) by lanthanum ions/atoms which - finally - could be visualized in TEM by Lanthanum's (atomic number 57) high e-dense contrast within the tissue. Lanthanum ions are able to bind to Calcium-binding sites and are able to replace calcium in a series of reactions (cf. e. g. BIRNBAUM et al. 1970; EVANS, 1990). Besides that properties there are calcium specific binding sites which can / will not react with lanthanum (an example is Concanavalin A).
Unhappy about perhaps no real use for /in histological preparation but it might be worth to give it at least a try (after having made a thorough recherche about technical possibilities to e.g. demonstrate substitution of Ca++ by La+++ also histochemically (specific stain).
*) La-chloride might be used too.
For convenience only (naturally there are far more and also more specific articles about that interesting chemical properties):
BIRNBAUM, E. R., GOMEZ, J. E., DARNALL, W. (1970)
Rare earth metal ions as probes of electrostatic binding sites in proteins
J Am Chem Soc, 92: 5287-5288
EVANS, C. H. (1983)
Interesting and useful biochemical properties of lanthanides
Trends in Biochemical Sciences 8(12): 445 – 449
(For more details I have to dig in my technical methods & processing copies and to search more recent, perhaps new literature)
You might have also a short look into:
‚Endochondral bone formation is involved in media calcification in rats and in men‘ , by: E Neven, S Dauwe, ME De Broe, PC D’Haese and V Persy (2007),
Kidney International (2007) 72, 574–581; doi:10.1038/sj.ki.5002353;
published online 30 May 2007, cf:
http://www.kidney-international.org/article/S0085-2538(15)52691-4/pdf
Thank you Wolfgang for your comprehensive answer. I will work my way through the points you mention. The more background I have, the better!
You're welcome, dear Robert. In case there is some detail left (uncertain) please: let me know or request 'elucidation' (:-)) thank you and best wishes and greetings to Dublin, Wolfgang
(PS: not only if you're considering "plastic"-processing I certainly could help anyway with protocols...)...
Dear Matthew, great contribution....(just to declare that the HNO3 I mentioned in my post / reply as decalcifying agent only was mentioned for "historical and general" reason...(:-))
NB: Regarding my statement in Re# 03: "Unhappy about perhaps no real use for /in histological preparation but it might be worth to give it at least a try (after having made a thorough recherche about technical possibilities to e. g. demonstrate substitution of Ca++ by La+++ also histochemically (specific stain) ",
I would like to inform that there IS a possibility to stain more/less specifically 'lanthanum-containing' (=substituted Ca++) tissue histochemically (i have found at least ONE specific reference/article).
Please Robert, do NOT decalcify your specimens. The mineral/soft tissue relationships are critical for understanding fully the phenomena you seem to be wanting to investigate. Many laboratories around the world are fully enabled to embed and prepare polymethylmethacrylate resin histological thin sections. This is routine in our laboratory, for sections ranging from ca. 5-100 microns. In addition, the sections can be imaged by any form of light microscopy, FTIR, and by scanning electron microscopy (SEM), and the mineral phase is conveniently imaged in the SEM using a common backscattered electron detector. If you would like references on methods, send me a message.
Dear,
I think Na-EDTA or formic acid might be good option to soften the calcified tissues like bones.
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