Click-iT™ EdU Alexa Fluor™ 647 HCS Assay

One may store the sample after fixation overnight in PBS at 4^oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.

Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.

The problem is likely not the Alexa Fluor 594 azide. Since there are no alkynes endogenous to mouse tissue, there is nothing for the dye-azide to bind to. Since the background doesn't overlap with nuclei (DAPI signal), this isn't an issue of unintended EdU labeling. This red is autofluorescence from red blood cells; they autofluoresce in the red and don't have nuclei. This can be confirmed by checking a completely unlabeled tissue section (no dye present at all) to see if they are still present and by examining the cells at high magnification and looking for corpuscular shape.

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Yes, but the standalone products are not shipped at the same amount as provided in the Click-iT EdU kits; the amount of dye-azide provided in the Click-iT kits is proprietary information. See these catalog numbers for the stand alone products:
- Cat. No. A10266: Alexa Fluor 488 azide
- Cat. No. A10270: Alexa Fluor 594 azide
- Cat. No. A10277: Alexa Fluor 647 azide

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The click reaction is only effective when copper is in the appropriate valency. Azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the TdT enzyme and click reagents to have access to the nucleus. Tissue samples require digestion with proteinase K or other proteolytic enzymes for sufficient TdT access.
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be o xidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Your cells may not be apoptotic. Prepare a DNase I-treated positive control to verify that the TdT enzymatic reaction and click labeling reaction are working correctly.

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The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You should also perform the complete click reaction on a no-TdT enzyme control sample to verify the specificity of the click reaction signal.

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