ab25374 staining the cortex of e14.5 mouse embryo by IHC-Fr.  The tissue was frozen and cut into 10 µm slices on a cyrostat then fixed and blocked prior to incubating with the primary antibody (diluted 1/100) for 90 minutes.  A fluorescent conjugated secondary antibody was incubated with the sample for 30 minutes.  HES-5 is shown in red in the nuclear regions; the blue staining is Hoechst nuclear counter stain.