In Situ Cell Death Detection Kit, Fluorescein

TUNEL assay cell type - Mouse skeletal muscle cells

Experiment
TUNEL assay cell type - Mouse skeletal muscle cells
Product
In Situ Cell Death Detection Kit, Fluorescein from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

Upstream tips
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

Total protein was isolated from cells by the RIPA buffer. Protein concentration was determined by the BCA (Pierce). Twenty micrograms of total proteins were resolved using the NuPAGE precast gels (Invitrogen) and transferred to polyvinylidene difluoride membrane. Following blocking with 5% BSA in TBS-T, the membrane was incubated with primary antibodies and secondary antibodies. Immunoreactivity was detected using Amersham ECL Prime (GE). The immunoblots shown represent the results of least three independent experiments (Supplementary Fig. 11). Protein density was measured with ImageJ.

Tissue samples were collected and snap-frozen in isopentane (VWR) liquid nitrogen slurry. For caspase 3 and TUNEL staining, limbs were placed in 4% formaldehyde for 1 h before being frozen. Sections (6–8 μm) were thawed at room temperature and fixed in 4% formaldehyde for 5 min. Sections were blocked with 10% donkey serum in 0.1% Triton-X for 30 min at room temperature. Sections were incubated with a relevant primary antibody overnight at 4 °C and with secondary antibodies for 1 h at room temperature. Antibodies were diluted in blocking buffer and sections were mounted in mounting medium (DakoCytomation) containing 5 μg ml−1 4,6-diamidino-2-phenylindole (Sigma). Myoblasts and myotubes were fixed with 4% formaldehyde or ice-cold methanol for 5 min. Cultured cells were permeablized with 0.5% triton-X for 10 min and blocked with 10% donkey serum, and follow the same staining procedure as tissue sections.

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Discussion

Discussion

2 years ago

Author: Ercole Udinesi Italy

Are 10 week old samples in formalin still usable?

I have left my tumor samples in formalin for an extended period of time (around 10 weeks). Do you think I will be able to use them for TUNEL assay and get results? Thank you for your help in advance

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for In Situ Cell Death Detection Kit, Fluorescein below.

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