Dear Anisa Annur Saidin,

You have not mentioned the source of embryo (is it from mammals or some other animals?). If you are interested to analyze the apoptosis in mammalian embryo, then I would like to give you some scientific and technical information that might be useful.

As you know that during early embryonic development, elimination of embryonic cell by apoptosis is an important way to eliminate the abnormal/defective or surplus/unwanted/damaged cells in order to ensure the proper development of healthy embryo.

Although Antczak and Van Blerkom (1999) reported negligible TUNEL

labelling in fragmented non-arrested embryos between the

two- and eight-cell stages. However, Yang et al. (1998) observed TUNEL-labelled nuclei in 16/21 fragmented embryos before the eight-cell stage.

Hence, under natural conditions, it is possible to detect apoptotic cells after fertilization and this is supported by the observations that

among embryos 7 days after IVF, those of 8 cells to morula stage presented a higher incidence of apoptotsis compared with the blastocyst stage in human (Read the review of Hardy, 1999; Reviews of Reproduction (1999) 4, 125–134).

The cytoplasmic fragmentation, developmental arrest, and nuclear condensation are the typical characteristics of embryos undergoing apoptosis. The TUNEL assay can be used as a way of selecting different grades of embryos and evaluating the quality of blastocysts. However, you have to be very careful for accurately analyzing the apoptotic cells from 8 cell embryo as the observation may caution in the interpretation of TUNEL results.

As you know that DNA strand breaks may occasionally occur as artifacts of fixation and specimen preparation (Collins et al., 1997). In addition to labelling apoptotic nuclei, positive TUNEL labelling has also been observed in cells undergoing necrotic cell death (Bicknell and Cohen, 1995; Grasl-Kraupp et al., 1995). Thus, it is important to confirm apoptosis using other morphological markers such as nuclear morphology.

In necrotic cells, the nature of the TUNEL labelling is different: the signal is not confined to the nuclei but is seen as diffuse staining in the cytoplasm as well (Charriaut-Marlangue and Ben Ari, 1995; Jurisicova et al., 1996).

The morphological features of apoptosis are not always associated with DNA fragmentation (Cohen et al., 1992). Fragmented nuclei without a positive TUNEL signal occur in human blastocysts. Conversely, TUNEL-positive nuclei are sometimes observed in the absence of morphological features of apoptosis (Sanders and Wride, 1996), indicating that DNA fragmentation is an early event in apoptosis. When TUNEL labelling is observed in arrested embryos, it is possible that DNA degradation is occurring in cells undergoing the early stages of secondary necrosis, rather than apoptosis.

Therefore, the Elabscience®TUNEL In Situ Apoptosis Kit (HRP-DAB Method) will stain nuclei of apoptotic cells (DAB brown color) and may also show diffused cytoplamsic stain in necrotic cells but not in non-apoptotic or healthy cells of 8 cell embryos. Thus, take utmost precaution while analyzing TUNEL results.

Best

Shail K Chaube

Shail K Chaube the source of embryo was from balb/c mouse