I am working on immunohistochemistry in testis tissue. After fixation in Bouin's fixative how long we can store testis tissue in 70% alcohol, before processing for paraffin embedding.
Hi dear Sunil KUMAR Patil,
In my experience, in IHC, 24-48h fixation in cold para-formaldehyde (4%) in 0.1M PBS buffer pH 7.6 has better results than Bouin. Just after 1h of fixation you should encapsulate testes to more penetration of fixative solution to be achieved. You can dehydrate your specimens :25%/50%/70%(3 changes)and 100%(2 changes) ethanol(30 min each) then xylene.That work very well . You can store your samples in 70% alcohol for long time (more than a month!) However; it is not recommended.
You can read an article (see below link) about Bouin and Davidson fixative solutions that may be interesting for you.
Good luck,
http://tpx.sagepub.com/content/30/4/524.long
Just my proposal:
find some posts regarding Davidson's fixative via RG-search-function, eventually pasting (when logged in to ResearchGate):
https://www.researchgate.net/search.Search.html?query=Davidson%20%20and%20fixative&type=question
Dito with:
https://www.researchgate.net/search.Search.html?query=Bouin%20%20and%20fixative&type=question
To answer your specific request ("how long can we store testis tissue in 70% alcohol, before processing for paraffin embedding - after Bouin's fixative"): I am not aware of a special reference or research article/work testing the efficiency (sensitivity and specificity) of IHC-staining of paraffin embedded material / sections after long storageof tissue blocks in 70% EtOH. Knowing that the removal of unbound picric acid (= yellow "bleeding" of the tissue blocks in 70% EtOH washes after proper fixation) usually can be stopped /is finished when the 70%EtOH-washing solution stays clear I remember from my personal experience that even if you had "clear solution after washing 3 x 1-2 hrs in 70% EtOH and got clear solution, you ended up with yellow solution in the morning again when one put that tissue blocks again in pure 70%EtOH, put the vials into a specimen rotator or shaker and left overnight in the fridge or at RT. So I guess that you might leave your testis specimens after Bouins Fixative quite long in 70% EtOH before further processing into paraffin and finally IHC on deparaffinized sections. IMHO your results will be affected / influenced more by your choice of a / the primary fixative , the pretreatment (e.g. preparation) and size of your specimens, the duration of fixation rather than washing and/or storage time in 70% EtOH, and - last but not least - the properties of proteins/substrata you want to localize immunohistochemically. Perhaps it could be beneficial for the community and the sake of science if you included a test for hypothesis (duration of thorough washing in 70%EtOH of your specimens after Bouin's fixative has no / has minor/major impact on IHC staining results). Best wishes and regards & good luck, WM.
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