FragEL™ DNA Fragmentation Detection Kit, Colorimetric - TdT Enzyme

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	For the in vivo apoptosis assay, eutopic endometrial tissue sections were fixed in 4% paraformaldehyde for 24 h at 4°C, washed in phosphate-buffered saline (PBS), and embedded in paraffin blocks. The tissue sections were incubated with proteinase K (20 μg/mL, without DNAase), washed three times with HBSS, and subsequently labeled using the TdT-FragEL™ DNA fragmentation detection kit (Calbiochem), according to the manufacturer’s instructions.

Protocol tips

For the in vivo apoptosis assay, eutopic endometrial tissue sections were fixed in 4% paraformaldehyde for 24 h at 4°C, washed in phosphate-buffered saline (PBS), and embedded in paraffin blocks. The tissue sections were incubated with proteinase K (20 μg/mL, without DNAase), washed three times with HBSS, and subsequently labeled using the TdT-FragEL™ DNA fragmentation detection kit (Calbiochem), according to the manufacturer’s instructions.

Publication protocol

For the in vivo apoptosis assay, eutopic endometrial tissue sections were fixed in 4% paraformaldehyde for 24 h at 4°C, washed in phosphate-buffered saline (PBS), and embedded in paraffin blocks. The tissue sections were incubated with proteinase K (20 μg/mL, without DNAase), washed three times with HBSS, and subsequently labeled using the TdT-FragEL™ DNA fragmentation detection kit (Calbiochem), according to the manufacturer’s instructions. As a negative control, Tris-buffered saline (TBS) was substituted for the TdT enzyme. Each section was analyzed in at least 3 continuous fields of vision at high magnification, and the number of apoptotic cells per 1,000 cells in each field was recorded. The TUNEL-positive cells were counted from more than 10 areas and averaged in each anatomic region using high-power-field (HPF, ×200) light microscopy.

For the in vitro apoptosis assay, the percentage of apoptotic cells was also assessed through TUNEL in endometrial cultures under basal conditions and after exposure to Leuprolide Acetate (LA) (25, 50, and 100 ng/mL).

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