Hi all, I am trying to stain the inner cell mass (ICM) of fixed human embryos with TUNEL to detect apoptosis (In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich) for confocal microscopy analysis. However, I only get to label trophectoderm (TE) cells. In my view, it seems that TUNEL cannot penetrate to the core of the embryo, and even after incubating embryos with 0.05 U/ul DNAse (positive control for apoptosis) only all TE cells are labeled but not ICM cells. Has anyone experienced similar problems before?

I perform fixation in 4% PFA/PBS for 20 min at RT, permeabilization in 0.5% Triton-X100/PBS for 30 min at RT, incubation with my primary and secondary antibodies overnight at 4ºC and 1h at RT, respectively; TUNEL incubation for 1h at 37ºC in humid conditions (9:1 TLM/TE) and DAPI staining for 15 min at RT. Several washing steps of 2 min in 0.1% PBST between markers.

Any kind of comment, suggestion or advice would be very much appreciated.