I've studied and fabricated a nanostructured substrate for the enhancement of neuronal differentiation.
I usually confirmed the levels of differentiation using immunocytochemistry, but I recently confirmed that a big problem occurred with the staining results.
The cells (NE4C) weren't stained with antibodies (MAP2), in which the results are attached to file 1. However, before I found the problem, there was no problem with staining, in which the results are attached to file 2.
Besides, I already tried to change the concentration of Ab working solution and incubation time...and use new Ab stock..., but I couldn't fix it. Please HELP me...
The ICC protocol I used is as follows.
1. 4% paraformaldehyde fixation for 15~20min
2. Permeabilization with 3% TritonX-100 for 5min
3. Staining with primary Antibody (overnight at 4 °C or 3 hours at RT
4. Staining with Secondary Antibody (1hour at RT)
5. DAPI staining
6. Treatment with mountain solution
As far as I can tell, the staining looks perfectly fine - I think in picture 2 the green signal is just more saturated.
How confident are you that the issue is with the staining and not the actual biology? Especially if you've already tried so many different variants of the staining protocol, it sounds very likely that the staining is showing you exactly what it should, and that it's the culture itself that's doing something different than before.
I agree with Benedikt Best ., looks like that is your result.
I would only recommend to do permeabilization softer, in 0.3% triton-X for 15 min, but if that previously gave you so nice staining, I have doubts about such changing.
Very nice pictures!
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