Did you have a positive control for your antibody? I wonder if your perinuclear signal is non-specific. Which antibody were you using?

Yes, we tested antibody specifity by peptide, and a lab in Japan had the same results like us. We used Mouse anti NeuroD IgM by abcam, Cambridge, UK and the lab in Japan used Rabbit anti NeuroD IgG also by abcam. The Rabbit IgG antibody does not work as nicely as the Mouse IgM, we used, so it is not clear, if the lab in Japan actually got signals in nuclei or not, but they did get a perinuclear cytoplasmatic signal. How about other transcription factors in postnatal and adult embryogenesis like DCX, TBR-1, TBR-2 etc? Any idea? Thank you so much!

Did you perform an antigen retrieval step in your immuno? eg. heating with citrate. This step is often needed when dealing with nuclear antigens. As for other "markers": Sox2 and Mash1 are frequently used. Dcx should work but won't be nuclear. Alternatively, Ki67 and PCNA can be used to show proliferation in the niche.

Yes, also did that, what we call demasking. Since NeuroD has been reported to be involved in the maturing process from type 3 cells on (after type 2b cells leave the cellcycle induced by Ngn-2 and NeuroD) in postnatal differentiation of glia cells, we would have expected NeuroD to be involved in adult neurogenesis also in the nuclei. Now we start to wonder, how different postnatal and adult neurogenesis are - cytoplasmatic and in the nuclei. Does Dcx show up at the same stages with same signal intensity in both for example? Maybe to compare postnatal and adult neurogenesis could be a topic for a PhD student. Any more ideas? Again, many thanks!

Very interesting! Does Dcx show up inadult perilesional neurogenesis in the same stages as in postnatal neurogenesis or in different maturity stages? However, in trauma there are more factors involved. For instance, after Status epilepticus NeuroD2 is upregulated, which is not the case in normal neurogenesis, postnatal or adult. Maybe this could be another project for a PhD student, to compare perilesional neurogenesis to normal one, postnatal and adult! Thanks!

These publications have nice NeuroD signal in the adult DG. Might be worth contacting the authors. PMID 19701198, PMID 19701197. It's difficult to appropriately quantify fluorescence across different tissues, because many factors can influence signal intensity. You would have to try to harvest the tissues at exactly the same time so that they can go through the fixation, cryoprotection, sectioning and immunofluorescence stages together. Unfortunately, immunofluorescence isn't really quantitative so it might be hard to legitimately make statements about signal intensity. You could certainly look into timelines etc though.

Thank you very much for the 2 papers, these are really helpful! Yes, for quantification western blot of course is more efficient, but immunfluorescence gives a more precise idea for cell localization. We did timelines and densitometric analysis at CT times. One paper used goat antibody to NeuroD (1:100, Santa Cruz Biotechnology), that would be worth trying. I am wondering, if signal location (nuclei yes/no) could be related to features of the antibody? This was the case about studies on malformation/hypoplasia of the dentate gyrus. A group had reported that no dentate gyrus was formed, while another detected a loose conglomeration. In ghrelin for example it is important to know, which subtype to use, and provided all these antobodies in NeuroD pass the peptide specifiency test, could that be somehow possible? Again, thank you very much for your great help!

Different antibodies raised against the same protein can give strikingly different results and require strikingly different treatment. I find PMID 12949777 to be a great guide to the voodoo of immunohistochemistry.

Thank you so much, I also forwarded it to my former PI, with whom I'm finishing up some writing right now. This is indeed a great guide for anybody doing immunohistochemistry! Thousand thanks!