Evaluation of anionic histological dyes as co-injectable cell markers in pre-implantation mouse embryos

The enzyme horseradish peroxidase (HRP) is a widely used microinjectable cell marker for studying cell position, lineage, and migration in many kinds of animal embryos. Marked cells are easily identified because they darken when exposed to a chromophore and an HRP substrate such as hydrogen peroxide. This assay, however, requires cytochemical fixation. Thus, when HRP-marked cells need to be identified prior to fixation, visible co-injectants such as dyes and fluorescent substances have been used with HRP. Fluorescent substances have limitations because their excitation could be harmful to the marked cells. Visible but non-fluorescent co-injectants, however, would permit visualization of HRP-marked cells without inflicting such damage. We tested the compatibility of several histological dyes and electrolytic carriers with HRP iontophoresed as a cell marker in 2-cell mouse embryos. The dyes tested were Evans Blue, Cibacron Blue F3GA, Fast Green FCF, and Patent Blue Violet; the electrolytic carriers were KCl, K2SO4, CH3CO2K, and KH2PO4. The combination found most useful was Patent Blue Violet in K2SO4. Survival of embryos incubated to the blastocyst stage following injection with HRP + Patent Blue Violet in K2SO4 at the 2-cell stage was significantly greater than that of embryos injected with any other dye. Although the proportion of embryos undergoing the 8-cell-to-morula transition was somewhat decreased by this treatment, the proportion of embryos reaching the blastocyst stage was comparable to that in the uninjected (control) group. Our results indicate that Patent Blue Violet is a useful, HRP-co-injectable dye for short-term cell marking in preimplantation mouse embryos.