I have found many different protocols to see amyloid-beta plaques using Thioflavine S and more or less all of them have the same steps: deparaffinization/hydration, incubate with 1% Thioflavine S (10'), ethanol wash (80-95%) and mounting media. It seems quite easy but I have tried and it doesn't work...I can't see any plaque in a mouse model which is suposed to develope them.
Have you considered the possibilty that there are no plaques in the tissues you stained, have you tried using another basic histological stain like congo red. Lastly have you considered having some one else (an independent observer) look at the slides if you are convinced that there shoild be amyloid plaques in the tissue specimen
For thioflavin staining, I have never dehydrated my tissue after staining. I stain my sections with thioflavin (0.1g/10mL), then to quench lipofuscin autofluorescence, the sections are incubated in 0.1% Sudan Black for 30 min, rinsed with PBS and cover slipped using PBS-glycerol (1:1).
Thioflavin stain is indeed very easy to look at. But if you are not seeing it, you can immunostain your tissue with 4G8 antibody to look at the presence of Abeta first. I know the purpose of looking at plaques with thioflavin is to avoid immunostianing, but with 4G8, you will know if there is indeed ABeta and Abeta-associated plaques are present in your tissue.
Thanks Adejoke! Congo Red was one of my options but I have not tried it yet, so I'll do it in the following days to see if there are plaques (yes I use independent observers but they are not experts in the field).
Thanks Gunjan! I will try the protocol you mention with Thioflavine. How many time do you leave the Thioflavine stain? You immerse the sample in the Thioflavine solution or you just add some drops on the top of the preparation?
On the other hand, I have used the 6E10 and there is Abeta, but I cannot see plaques (at least not the ones that appear in the majority of publications) maybe because of my lack of experience or maybe because the antibody is not specific enough...I don't know.
Have you considered that you animal has plaques but they are not reactive to ThS? It might be that the plaques are not compact enough since ThS stains for fibrillar Abeta. What if you have diffuse ones? Have you tried with an antibody against Abeta? Don't you have a positive control? a human AD sample? In addition, most of the times, DAPI (the nuclear staining) labels the same kind of plaques than ThS, you may want to try it just in case. Plus, you can dehydrate the samples after ThS, there is no problem with that.
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