Hi; My name is Oscar and I´am a Lab Technician at the Neuroscience institute of the Autonomous University of Barcelona; thanks in advance for reading.
I´am actually working on the Simplified Gallyas method, we want to impregnate TAU tangles with the silver precipitate. Find the protocol attached.
I´have some doubts:
I´have achieved the impregnation in human samples nor in mice transgenic mice.
I´am using DPX as mounting medium, the protocol stands for cytoseal but as the clearance steps are the same as DPX and this is the medium we use in our lab most... I decided to use DPX so... ¿Can DPX modify in a way or another the impregnation? I can see differences on the staining (from stained tangles one day to nothing stained the day before in the same slide) and I don´t know if it can be for the DPX or for the staining itself or maybe I am doing something wrong.
Solution expiry dates?
Storage particularities of samples or solutions?
Any advice?
It´s my very first time I´m trying that, so any support will be precious.
Thank you very much.
Hi Oscar,
I think you should not worry for the DPX, it's a bit old in the formulation, so you might want to switch to Entellan, but all these embedding resins are based on polystirenes and use toluene as a solvent and would not affect metal deposits.
I think it is a general problem of silver stainings the difficulty to obtain uniform staining. That's why most protocols have developed into exchanging the silver with the gold (the new Golgi stain formulations, Black Gold II, etc). I see that your Gallyas is not using this trick, which is normally used in the modified or simplified Gallyas methods, although it makes it substantially more expensive... Maybe you wish to have a look at this paper:
Article Simplification of the modified Gallyas method
Good luck.
Thanks Pietro for your answer;
Yes, we avoided some steps trying to make our staining reproducible & cheaper, but I think that combination of terms would not result in this case.
Do you know any other method (not immuno) to stain TAU tangles?
I think I would try the expensive/modified/simplified version at last...
Thank you very much.
Well, there is also the possibility to use Thioflavin S, which is a fluorescent dye that stains beta pleated sheets, so both NFT and b-amyloid plaques, which you can then distinguish by shape, size and location. There is another much older thread in ResearchGate:
https://www.researchgate.net/post/Which_AD_hallmarks_does_Thioflavin_S_label_Amyloid-plaques_or_Neurofibrilary_tangles_Or_both
I was looking myself into these approaches for a possible phenotype in our mouse model, but in the end I never started the project because we don't have grounds at the moment to think we have a tau pathology, so I don't have direct practical experience, only a bit of studying...!
Yes, thank you Pietro.
At this point I don't know if we want to stain exclusively TAU, I suppose so...
Anyway, I also learned from you the use of Thioflavine S that is the cheapest alternative on the purpose, sure will be helpfull in the future.
Thanks again Pietro!
thanks for all your support!!!
I have positive satining in human and Mice.
Best regards.
Oscar.
I think that the potassium permanganate (for background) the gold chloride (for cleaning unspecific staining) and the sodium thiosulfate (to fix the stain) steps where very necessary...
I followed the Nobuo Kuninaka "modified" stain scrupulously and that´s all, simply not trying to make it cheaper this time.
take care Pietro!
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