Click-iT™ EdU Imaging Kit with Alexa Fluor™ 488, 594, and 647 Azides

この問題は Alexa Fluor® 594 azide の染色ではないと考えられます。 マウス組織には内在的なアルキンはないため、Alexa Fluor® 594 azideが結合するものはありません。 また、この染色が核染色 (DAPI) とも重ならないため、これはEdUの非特異染色とも異なります。 これは、実際には赤血球が見えています。赤血球は赤い自家蛍光をもつ一方で核を有していません。 これは完全に未標識(色素も添加していない）切片でもこれらの染色が存在するかどうか確認することで検証できます。 また、高倍率の観察で赤血球の形状も見られます。

Click reactions have several general characteristics: the reaction is efficient, no extreme temperatures or solvents are required, the reaction is complete within 30 minutes, the components of the reaction are bio-inert, and perhaps most importantly, no side reactions occur-the label and detection tags react selectively and specifically with one another. This final point is a key advantage of this powerful detection technique; it is possible to apply click chemistry-labeled molecules to complex biological samples and detect them with unprecedented sensitivity due to the extremely low background of the reaction.

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

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.

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

We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.

For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.

Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.

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