Hi all,
I will be doing some staining on a whole nerve and subject this to x ray microtomography.
Has anyone ever used Tannic acid for this technique? If so, what's the concentration of the acid? 1%? 2%?
Thank you
Dear Laura, I'm a bit confused about the issue you are trying to solve:
i) knowing a bit what x-ray microtomography means
ii) knowing a bit about "histological" as well as whole tissue/organ staining
iii) knowing a bit about use of Tannic Acid (TA) in Histology, Light microscopy and Electron Microscopy
iv) knowing therefore a bit about use of TA either in combination with the secondary fixative OsO4 OR the application of TA AFTER a secondary fixation of tissue with OsO4
v) Guessing you have 'prepared"= dissected a nerve or at least part of it out from the (which nerve, animal?) body and
vi) would like to "image" that whole nerve specimen by means of X-ray Microtomography
So my first question(s) is/are:
- whole nerve specimen fixed or unfixed?
- if fixed: which fixative and when?
- which size?
- any experimental treatment one should know about to decide how to proceed further?
- has - for any reason - the native nerve material to be 'left native' or can it be "fixed"(in terms of 'chemically induced coagulation")?
- Do you have access to "harsh and hazardous chemicals" like Osmium Tetroxide (OsO4) which I only anticipate due to yyour demanding "Tannic acid" (which per se in higher concentrations might / (quoted from an MSDS)"...could cause potential health hazards such as damage to the eye, skin, respiratory tract, and gastrointestinal tract. It may cause irritation, redness, pain, blurred vision, and possible eye damage. When tannic acid is absorbed through the skin in harmful amounts, it may cause irritation, redness, and pain. Nausea, vomiting and diarrhoea are symptoms of tannic acid ingestion and prolonged exposure may cause liver damage. Upon inhalation, tannic acid may cause respiratory tract irritation ."
Succus:
If you CAN fix the whole nerve (as usual for Electron Microscopy = SEM or TEM) primarily either in situ (anatomically) or right after dissection from the original location I would try to
either:
i) mordant the nerve by immerging /incubating in 0.05-0.1% hydrous (or appropriately buffered) microfiltered TA-solution (use ultrasonic dissociation, use at least 0.46µm syringe filters; apply in the dark) for at least 1 - 3 hrs (4°C or @ RT) followed by (hydrous or appropriately buffered) 1-2% OsO4 solution (at least 1-2 hrs @RT), followed by multiple /ample washings with the vehicle buffer afterwards prior to X-ray microtomography)
ii) after primary fixation (usually FA or FA-GA-mixtures in suited buffer solution): Osmication (OsO4 admixed with Tannic acid: recipes are out in the wild, search eg. for OTO, or "OTOTO and mordanting"
[CAVE/CAUTION: use personal protective equipment, use and work only in a fume cupboard or well veentialted working area! Don't breathe in / inhale OsO4 vapors!]
- appropriate washings - dehydration starting with 50% EtOH, and finally -incubate nerve specimen in 1% PPD (=para-phenylenediamine) in 70% EtOH (approx. 1-2 hrs @RT), wash several times for 10-15 mins each (to get rid of unbound PPD) then image...
If you have any questions left on the "Methods" I proposed, please request more information directly sending an e-mail to womuss@gmailcom.
NB: I have no idea whether a TA-solution (ONLY) has been applied to incubate biological tissue to increase "contrast" of imaging by X-Ray-Microtomography... (but I know there exist X-Ray-Microtomographical studies to image tannin-containing materials, like foams....) . If anybody has knowledge about the Use of TA in that manner, I would love to read about....
As only ONE example of technique one could use:
CHENG et al, 2016: Research Article
Ultrastructural Mapping of the Zebrafish Gastrointestinal System as a Basis for Experimental Drug Studies
BioMed Research International Volume 2016, Article ID 8758460, 13 pages
http://dx.doi.org/10.1155/2016/8758460
[(C) Hindawi Publishing Corporation], can be found Open Access @
https://research-management.mq.edu.au/ws/portalfiles/portal/62321250
(X-ray-microCT on resin-embedded Zebrafish sections)
Personal Disclaimer: no warranty for easiness, reliability, or positive outcome/result (:-))
Best wishes and regards, as well as GOOD LUCK, W.M.
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