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I compared muscle stem cells from two metabolically distinct muscles – I found that these populations stained differently for Wnt2, with one population having more intense nuclear staining that the other. I have attached images for you to see the difference. In the image with the darkest staining (nuclear), you can see that there are also cells which are negative for the stain, which suggests that it not therefore indiscriminately staining all nuclei. There is some light staining of the cytoplasm also for both populations which I don’t know if this is as expected or due to too high Ab conc.
OK. I use the Vecta stain Elite ABC kit
- Grow cells as monolayer and fix in 70% ethanol, wash 2x pbs, 1x water, store in water until staining
- Block in 1% normal horse serum 30 mins
- Remove NHS and add primary antibody – Wnt2 (AB109222) 1/200 dilution in PBS (range on data sheet 1/100- 1/250)
- Inc 1 h at room Temp on rocker
- Wash 3x pbs
- Add secondary antibody – biotinylated anti-rabbit (1%), inc as before.
- Wash cells 3x pbs
- Add 1% A+B reagent as Vecta protocol states for 30 min
- Was cells 4x pbs
- Add substrate – peroxidase (VIP substrate kit) ˜5 mins
- Wash 3x pbs
- Wash 1x water, store in water.
I look forward to your comments.
Asked on Mar 01 2012
Thank you very much for providing the images and protocol info.
I have checked different publications and databases; all show cytoplasmic staining. The links to the few are attached below;
The following Abcam antibody also show cytoplasmic staining
I would suggest tweaking a protocol and then checking if the result improve
- Fix cell in acetone for 10 minutes, or 4% icecold PFA or icecold methanol
- Use 5 - 10% NHS for blocking (1hour)
- Try the normal ICC/IF procedure using fluorescent secondary antibody
- Use PBST (0.1% Tween) instead of PBS
-Try no primary control also
The other question I have is
- Have you stained nucleus by any DNA dye?
I hope the results will improve after following these suggestions. Please let me know if you have any feedback or would like to discuss this further.
Answered on Mar 01 2012