I have tried conducting iba1 and GFAP staining in 40um sliced rat brain sections. The staining seems to work well on the peripheral parts of the brain but not the central. There are the occasional cells that show a strong signal, but the rest of them do not. This leads me to think those peripheral parts which will have had the strongest penetrence of the PFA will be the ones that have fixed the strongest.
I have slices from rats which underwent a very similar protocol but I conducted this months before and the IHC protocol works fine for those ones.
I am not 100% sure that it is under fixation. But I can not think of what else it could be. I believe over fixation show very strong positive staining and noise. Also why would over fixation only show strong signal in the peripheral parts of the tissue.
I enclose a not very good image of this currently. But it highlights my point, the right of the image is the periphery, where there is strong staining. The left is going more medial.
Is there anything that I can do to try to understand what has happened, and what I could do to try to get usable images from this tissue?
Hi, depending on your fixative, I remember that the rule for the penetration time is around 1 mm/h. FYI: https://www.rcpa.edu.au/getattachment/d6f7f095-e8b7-45eb-8dcb-6a9d9bd5a88a/Fixation-of-Tissues.aspx.
- Your hypothesis seems logical to me - Have you tried postfixation on the slide?
- Was it freshly prepared PFA or did it stand for a longer time in the lab?
- If the PFA was fresh, a fixation for 24 hours should be sufficient due to the size of the sample and I don't expect a difference in the fixation between central and peripheral parts.
I agree with Erdem Yildiz and I would add another question, did you trans-cardiac perfuse with PFA? Immersion fixation of brain tissues can be problematic. Typically, we perfuse with 0.1M PBS to flush blood out followed by 4% PFA. The rate of flow is also important, with adult rats 200mL of PBS over 4min and 350mL over 8min of PFA (slower PFA the better). Additionally, we will immersion fix the whole head for 24-72hrs, then extract brain from the calvaria and begin cryoprotection procedures. Essentially, this covers two fronts movement of the fixative from inside and outside the tissue. In some cases immersion fixation can isolate the interior of the brain tissue as the osmotic flow is reduced (time negates this but over-fixation and crosslinked/ damaged antigen are the result). Hence, fresh fixative is best.
Finally, it is hard to identify true peripheral vs central from the image provided. If the image appears as a bright ring of staining surrounding an area of little or no staining this may suggest artifact of some kind. It may be that the edges of the tissue were damaged due to freezing/ thawing too rapidly, e.g. -80C to room temp OCT. In this case the areas in the center of the tissue are correctly stained and the periphery is incorrect.
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