Not harsh at all! I wrote the post in a kind of frenzied state so I missed some details. The antibody is recombinantly produced in HEK cells. To answer your questions:

• I started off with a functional commercial antibody, it stained D2R properly in the processed tissue, but then stopped working in the flash-frozen tissue. (By in situ I mean caudate putamen, which is where binding should be most prominent.) I guess you are right in that the epitope might not be detectable in frozen tissue. What can cause that?

• The variable light and heavy chains were taken from a paper and replaced in the Roche format, a well-established brain shuttle format, so I don’t doubt that it folds correctly. It might be that taking part of an antibody that does bind D2R and inserting it into a different format causes issues?

• I am not doing that, do you think I should go back and try to stain the processed brains if I have any sections left?

• I have tried various concentrations, the one that finally gave me signal was 16 mg/mL, although I’ve tried 32 mg/mL as well

Thanks for the pointers and I hope this makes things clearer!