I recently received postmortem (aged) human brain samples in a cassette (previously fresh frozen) from brain bank.
I would like to use those samples for fluorescent immunostaining.
Could anyone suggest me any articles that I can refer to?
I would like to make sure the % of PFA and duration of fixation for the tissue preparation before proceeding to cryopreservation and sectioning (cryostat).
Kind regards,
Some parameters necessary to observe with such an attept are depending on further, more detailed information on steps
i) "quality" of primary freezing the hum. brain specimens (which means also: how long was the time passed until p.m.-excision, how brain sample [size] was frozen [technique], storage in frozen state / 'in cassette'), process of thawing?, etc).
Generally - I 'guess' - you should adhere to all those protocols availabe by primary Literature Res.or as outlined in several "handbooks" of Histology, IHC, ICC etc. (e.g. only one source out of many to mention here:
Bancroft's Theory and Practice of Histological Techniques
8th Edition 2019 ( S. Kim Suvarna, Christopher Layton and John D. Bancroft, Eds.)
https://www.elsevier.com/books/bancrofts-theory-and-practice-of-histological-techniques/unknown/978-0-7020-6886-7, or,
https://www.sciencedirect.com/book/9780702068874/bancrofts-theory-and-practice-of-histological-techniques, respectively.
Last but not least: please specify more precisely what you are going to do really or plan to do:
Rwa data for now from you Q:
i) you got (previously frozen) human brain samples from a brain tissue bank -in a cassette [NB: now frozen or thawed?]
ii) you would like to process those samples in a manner to get sections which are to be immunostained /IF-stained ,
but
iii) you ' would like to make sure the % of PFA and duration of fixation for the tissue preparation before proceeding to cryopreservation and sectioning (cryostat). '
As of my restricted, 'dense' knowledge it might be appropriate to use either an appropriately! buffered, FRESHLY prepared 4% or even 2% (PFA) para-formaldehyde solution for fixation with a duration of (depending on sample "volume/thichkness/size") at least 2-5 hours at RT (perhaps also over night at 4°C).
If brain samples are p.m., and/or intermediately have been frozen/thawed/frozen/ etc. it may well be that the evaluated and assessed IF-staining results have to be judged critically...
Best wishes and good luck (hoping there will be other colleagues chiming in....)
Hi Yuka,
NBF fixated brain will present some autofluorescence issues in the yellow green range due to natural substances (lipofuschin, heme proteins, blood vessels, hormones, collagen etc.) due to the cross linking characteristics of aldehydes.
Here is a list to get you started:
https://bitesizebio.com/webinar/fundamentals-of-fluorescence-microscopy/
http://www.ncbi.nlm.nih.gov/pubmed/10330448
https://biotium.com/technology/primary-secondary-antibody-conjugates/considerations-for-immunofluorescence-staining/
https://biotium.com/support/tech-tips/tech-tip-battling-tissue-autofluorescence/
https://www.frontiersin.org/articles/10.3389/fnana.2019.00073/full
https://med.emory.edu/departments/medicine/divisions/cardiology/research/labs/microscopy-in-medicine/_documents/removal-autofluorescence-sudan-blackb.pdf
https://journals.sagepub.com/doi/full/10.1177/002215540104901210
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