Protocol for immunofluorescence detection and quantification of phosphorylated SMAD proteins in human blastocysts

The transforming growth factor β (TGF-β) signaling superfamily includes NODAL and bone morphogenetic protein (BMP) signaling, which lead to the phosphorylation of different SMAD proteins and regulate key developmental events. Here, we present a protocol for immunofluorescence detection of phosphorylated SMAD proteins combined with other transcription factors in pre-implantation human embryos. We describe steps for segmenting the nuclei in human blastocysts and quantifying their immunofluorescence intensity. This protocol can be adapted to investigate TGF-β superfamily signaling activity in other mammalian embryos or in vitro models of their development. For complete details on the use and execution of this protocol, please refer to Brumm et al.¹.