Dear colleagues,
As a neuroscientist and anatomist, I work with human brain material, which we gain in our dissection course. We use a mixture of alcohol and PFA for fixation. After brain removal we store the brain in 4 % PFA solution. However, we face the problems many in the field encounter that the pH is decreasing over months, after 1y, a pH of 5 is average.
Does anybody know a chemical procedure to make it longer stable?, to keep the physiological pH?
Thank you very much in advance!
Best,
Michael
The answer would depend on what would you like to do with the fixed brain tissue after storage - is it just for anatomical dissection or histological analysis?
There are different options (based on the answer) but they all have negatives:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2062777/pdf/amjpathol00719-0111.pdf
Article Practical Aspects of Neuropathology: A Technical Guide for W...
https://www.cambridge.org/core/services/aop-cambridge-core/content/view/D90F6330DFAA8CB0614EDBE7716C91A7/S1551929510000155a.pdf/why-4f-1g-fixative-works-for-me.pdf
I hope this helps :)
Dear Michael,
You might want to consider to use the Fix for Life (F4L) embalming and preservation method. The brains can be stored in F4L-immersion fluid (pH 7 ±1) which contains less than 0,2% formaldehyde (brain tissue will be just slightly softer, white and grey matter even better distinguishable). More info at: https://fixforlifeembalming.com
Kind regards, Dries
Thank you to your very helpful comments - all of them will be taken into consideration.
to Valeria: we want to make IHC with primarily synaptic markers - do you have any further ideas?
Thank you again! My first question here and I already feel the spirit of the community and help here:)
best, Michi
Well, I do have ideas, but do you have the funds to realize them? :)
In an ideal world, whereby university researchers get the best technology to work with - instead of distortion- and shrinkage-prone chemical fixation, I would have recommended the Japanese "cell alive" freezing method instead:
http://japan-product.com/abis-cells-alive-technology-cas/
alternatively,
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4530226/
Article Feasibility of high pressure freezing with freeze substituti...
CAS has been used to freeze and defrost meat and fish (i.e. high water and fat content) with excellent results for some years now, and although used commercially (and not for science), it would have been the best (and the least toxic) method for us to preserve and explore human tissue with.
Since the system is not used in anatomy or histology yet, all other options are a kind of a compromise.
Antigen retrieval methods, based on routine tissue fixation:
Article Reversing the Effects of Formalin Fixation with Citraconic A...
Article A New Antigen Retrieval Technique for Human Brain Tissue
Article Fixation Strategies For Retinal Immunohistochemistry
(evaluation of best bets)
Information related to your research:
Article Quantitative immunohistochemistry of synaptophysin in human ...
Chapter The Importance of Titrating Antibodies for Immunocytochemica...
Article Quantitative immunohistochemistry of synaptophysin in human ...
Article Measurement of pre- and post-synaptic proteins in cerebral c...
Article Quantitative immunohistochemistry of synaptophysin in human ...
(shorter tissue fixation though)A good source of additional information:
Corthell, J.T. "Basic Molecular Protocols in Neuroscience: Tips, Tricks, and Pitfalls" Chapter 9 - Perfusion and Immersion Fixation. Academic Press, 2014, pages 83-90.
I hope this gives you more precise options. :)
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