Incubation of Primary and Secondary Antibodies Help - Immunohistochemistry (Jul/30/2011 )
I have a question regarding how primary and secondary antibodies should be incubated. I have tried two methods so far and have gotten drastically different results and wonder why this is so.
At first, I tried simply diluting the primary antibodies in 1%BSA and then adding it to the samples in a petri dish. On the sides of the petri dish are wet kimwipe so that when I close the lid of the petri dish, the primary antibody solution does not get dried. I incubated the secondary antibody the same way. After mounting, and looking at the pictures, my signal was very weak and I could see very few stained tight junctions proteins (my target protein).
On my second attempt, I decided to add parafilm on top of the primary antibody solution to spread out and flatten the solution on top of the sample. After mounting this time, the signal was a lot stronger and a lot more stained tight junctions can be seen.
1) Does anyone know why the second method gave me better results? 2) Is there any better method for incubating antibodies that you guys know?
I have never heard of antibody staining done without some kind of coverslip (parafilm, in your case). I don't know why the first method wouldn't work.
Are your samples on a slide or on the petri dish itself? For samples grown on coverslips, I cut parafilm to fit inside a petri dish. Pipette antibody solution onto the parafilm (it will bead up) and gently place inverted coverslip w/sample onto the the solution (so sample is face down with a layer of solution between parafilm and coverslip. Petri dish was place in a humidity chamber (sealed plasticware with saturated paper towels/kimwipes).
I have also done this with samples on slides, adding solution to the slide and placing a coverslip on top of it. M-series Lifterslips work better that regular coverslips -- they allow for more solution and more movement of molecules/antibody in the solution.