You could try much longer incubation times. Some researchers in our
group incubate free floating sections in primary antibody for 2-3
days on a rotating table, but we cut at about 50µm max in our lab for
IHC. Have you tried Creysl violet staining and can you get good
penetration with that?


At 5:46 PM +0000 4/3/03, sue garcia wrote:
>Hi Histolanders,
>I have the following problem: I am trying to stain free-floating
>thick frozen sections for IHC using different antibodies.
>Independently of the tissue or antibody of choice, I can only get
>stain about 30 microns deep into the section, while the rest (about
>200 microns total) remains unstained. I have tried different
>permeabilization methods, but nothing seems to solve this problem.
>Lately, I have been doing 0.8% Triton X-100 in PBS overnight at room
>temperature, and sometimes I can get up to 40 microns deep...Any
>protocol out there for staining of free-floating tissue sections
>would be greatly appreciatted.
>Sue
>
>
>
>
>
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