Hello Megan Schupp

For better penetration of antibody, Triton X-100 is used. Triton X-100 can dissolve the lipid on cell membranes, nuclear membranes and organellar membranes, therefore big molecules such as antibodies can get into cytoplasm and nucleus to bind antigens. A 0.5-1.0% solution of Triton X-100 in PBS works well on tissue sections when applied before the primary antibody. Triton X-100 is not suitable using in antigen retrieving when known antigens are localized on the surface of cells because Triton X-100 destroys membrane structure.

I would suggest the most suitable way would be to start again without using Triton X-100 in the buffer. You may include Triton X-100 in the buffer during the permeabilization step.

Best.

I agree with Melcom, I used Triton - X just in my dilution media for the antibodies in a concentration of 0,2%. For the rinsing steps I use PBS without TX. If you are using the free floating technique you will receive good results with this protocol.