Hello, I am planning to stain retinal tissue sections for IHC and want to co-stain cones and rods. For this we plan on using a Lectin PNA From Arachis hypogaea (peanut), FITC Conjugate for cones and an uncojugated Rho antibody (rabbit) with a secondary fluorescent antibody (G-anti-rabbit, alexa 568) for rods.
My question is if it is possible to use both reagents in the same slide without any interference for double staining.
I was thinking of applying the antibodies first (1ary ON and secondary for 2 hours), wash the slides with PBST 0,1% and apply the lectin.
My biggest questioning is if the amplified signal from the antibodies would mask the lectin signals. Does anyone have experience with this? any reccomendations?
I did this before not it works well as you planned to. With unconjugated primary going first and then PNA lectin in the end you can also add DAPI to the lectin solution if you need a DAPI signal in your samples. I would rather use 647 as rods are usually quite auflourecent at Cy2-Cy3 channels.
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