ApopTag® Peroxidase In Situ Apoptosis Detection Kit

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	Cells were treated with 20 mg/ml proteinase K for 15 min at room temperature after after deparaffinization. - hydrogen peroxide is applied for 5 min at room temperature.

Protocol tips
Cells were treated with 20 mg/ml proteinase K for 15 min at room temperature after after deparaffinization. - hydrogen peroxide is applied for 5 min at room temperature.

Publication protocol

5-FU-induced cell death was investigated on the day 14 using the terminal deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labelling (TUNEL) method (ApopTag®, no. S7101; Merck Millipore, Darmstadt, Germany). Briefly, after deparaffinizing, the samples were rehydrated and incubated with 20 mg/ml proteinase K for 15 min at room temperature. Endogenous peroxidases were blocked by treatment with 3% (v/v) hydrogen peroxide in phosphate-buffered saline (PBS) for 5 min at room temperature. After washing, the sections were then incubated in a humidified chamber at 37°C for 1 h with TdT buffer containing TdT enzyme and reaction buffer. Specimens were incubated for 10 min at room temperature with a stop/wash buffer and then incubated in a humidified chamber for 30 min with anti-digoxigenin peroxidase conjugate at room temperature. After a series of PBS washes, the slides were covered with peroxidase substrate for color development and then washed in three changes of dH2O and counterstained in 0.5% (w/v) methyl green for 10 min at room temperature. The TUNEL-positive cells were counted (10 fields/slide; magnification, ×1,000) for statistical comparisons.

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Manufacturer protocol

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