In Situ Cell Death Detection Kit, Fluorescein

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Seed 10,000 cells in 96-well plate. Fix sample in 4% formaldehyde for 1 h before freezing.	Fix sample in 4% formaldehyde for 5 min before using sample. Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature. Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature

Upstream tips
Seed 10,000 cells in 96-well plate. Fix sample in 4% formaldehyde for 1 h before freezing.
Protocol tips
Fix sample in 4% formaldehyde for 5 min before using sample. Block samples in 10% donkey serum in 0.1% Triton-X for 30 min at room temperature. Incubate primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature

Publication protocol

The apoptotic cells were quantified for DNA fragmentation, by using an In-situ Cell Death Detection kit, Fluorescein (Roche Diagnostics GmbH, Mannheim, Germany), according to the manufacturer’s protocol. In brief, excised tumor tissues were fixed in 4% paraformaldehyde, dehydrated and embedded in paraffin. Paraffin-embedded tissues were cut into 5-μm thick sections. The sections were deparaffinized and rehydrated according to standard protocol. The tissue sections were then antigen retrieved at 0.1 M citrate buffer for ∼5 min in a pretreatment module (pH 6.0) and then rinsed twice in PBS. The slides were then treated with 50 μl of ‘TUNEL reaction mixture’ and incubated at 37 °C for 60 min The slides were then rinsed 3 × with PBS. Negative and positive slides were included. For negative slides, only 50 μl of ‘Label solution’ (without terminal transferase enzyme) was used and for positive slides, the slides were treated with DNase I before proceeding with treating with ‘TUNEL reaction mixture’. For microscopic image capture, four high-power fields were randomly selected and the apoptotic index was calculated as equal to number of positive cells/total number of cells × 100%.

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Manufacturer protocol

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