Hello, I am trying to study synapses and try to stain synaptophysin and synapsin on postmoterm rat brain tissue. But so far have completely no siangls under confocal (I am expecting to see punctas).
Rat brain tissue is kept in 4% paraformaldehyde for over 3 years.
My protocols invoves 10% serum for preblock and 1% serum for antibody incubation. PH7.4 PBS buffer washes between steps and 1 night first andtibody and 1 night second. Dilution factor range from 1:200-500 for primary and 1:200 for secondary. I tried antibodies from Milipore and Fisher as how other paper used them.
I also tried staining PV for positive control and I see perfect singals (so the tissue should be fine).
Do you have any suggestions where the problem should be? or any suggestions of change s in antibodies use or protocol changes?
https://www.sysy.com/product/101008
It could be the case especially this antibody does not recognice the synaptophysin eptopes after 3 years PFA fixation. if you are using paraffin section antigene retrievel is highly recommended. For frozen sectoins I recommend a dilution medium which contains not ony normal serum you should add Troton X 100 in a concentration between 0,2 and 0,5 %.
Don't use green fluophores (Cy2 or alexa 488) because of the long lasting fixation the unspecific background staing increases, especially in the green chanel. This could be one reason why you don't see anything, because the background has swallowed your signal.
It could be possible that your detection system is not sensitiv enough. Instead of labeld secondaries use biotinylated secondaries and detect them with labeled Steptavidin. If this is not enaough you can try to improve your results with tyramide signal amplification systems. Or you try a new antibody. I have given you a link to Synaptic Systems. This company offers alot of different synaptophsine antibodies. Good luck!
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