Click-iT™ TUNEL Colorimetric IHC Detection Kit - FAQs

View additional product information for Click-iT™ TUNEL Colorimetric IHC Detection Kit - FAQs (C10625)

9 product FAQs found

Can Metal Enhanced DAB Substrate Kit (Cat No. 34065) be used instead of the DAB provided in the Click-iT TUNEL Colorimetric IHC Detection Kit?

Yes, but we have not optimized its use with this kit.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

Can the Click-iT TUNEL Colorimetric IHC Detection Kit be used with other colorimetric dyes?

Yes, other colorimetric stains can be used.

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I ran out of the TdT enzyme used in the Click-iT TUNEL assay. Can I purchase it separately?

Yes, additional Terminal Deoxynucleotidyl Transferase (rTdT) can be purchased as Cat. No. 10533-065 or 10533-075. Additional TdT reaction buffer can be purchased as Cat. No. 16314-015.

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Can I perform Click-iT EdU TUNEL detection on cells growing in 3D culture?

We have not validated the use of EdU TUNEL for apoptosis detection in 3D culture systems, but as this reagent is compatible for labeling cells in vivo, it is also expected to label cells in 3D culture systems. There are a number of reports in the literature that use this product in 3D culture systems; here are some citations:

Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci U S A 110:E5039-E5048.
Derda R, Laromaine A, Mammoto A et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc Natl Acad Sci U S A 106:18457-18462.
Robertson FM, Ogasawara MA, Ye Z et al. (2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype. J Biomol Screen 15:820-829.

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For the Click-iT TUNEL for In Situ Apoptosis Detection Assay, how long should I digest my tissue samples in proteinase K?

Most tissue samples will be adequately digested in 15 minutes. The optimal incubation time will vary depending on tissue type and thickness. We have observed that brain tissue needs longer proteinase K treatment than other tissues tested.

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How thick can my tissue sections be for apoptosis detection using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay?

We have validated the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay on 5-20 µM thick FFPE sections of mouse intestine, kidney, liver, heart, and colon.

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Can I use the original Click-iT TUNEL assay kits on tissue samples?

The original Click-iT TUNEL assay kits were optimized for cell culture samples and may also be used on tissue samples. We recommend using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue. The protocol was modified to improve accessibility of the TdT enzyme into the multiple cell layers of tissue samples. In addition, we found that the original Click-iT TUNEL kit may show higher non-specific binding and punctate staining in tissues compared to the Click-iT Plus TUNEL kit. One method that works well to increase permeability for tissues is to replace the detergent permeabilization step with proteinase K digestion and then refix the sample in formaldehye. The Tissue Fixation and Permeabilization protocol in section 3 of the Click-iT Plus TUNEL kit manual can be followed for tissue samples using the original Click-iT TUNEL assay. Pepsin and other proteolytic enzymes can also be used to improve permeability.

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What are the advantages of flow cytometry?

-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.

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What kinds of applications can I run on a flow cytometer?

There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.

Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.