I've been working on differentiating Rat fetal Neural Stem cells into Neurons with a drug of interest. The Problem here for me is I have used various Neuronal markers like MAP2, TUJ1, NFM to differentiate the Neurons. But the Western blot results are confusing. I have to compare my result with an undifferentiated stem cell control and differentiated Neuron without my drug of interest. I an attaching the fig of my wb data. When I use the TUJ1 marker the stem cell also express it and the untreated shows higher expression when compared to my drug of choice. But the other two neuronal markers I have used MAP2 and NFM shows higher expression in my drug of interest. My other experiments shows my drug is working like the neurite counts was higher, length of the neurite was longer and more number of differentiated cells can be seen when I treat my drug. My ICC for TUJ1 clearly shows the neurite growth and branching is more significant in my drug and MAP2 shows a perfect well developed dendrite. My explanation for the WB data is that TUJ1 is a marker for immature neuron and once the Neurons gets develops it gets replaced by MAP2 as a cytoskeletal protein. That is why the TUJ1 expression in my untreated cells is higher than my drug of interest where as the expression of MAP2 and NFM are higher in my drug of interest because there are more mature neurons and their cytoskeletal protein TUJ1 was replaced by MAP2 and NFM. Is my explanation makes sense?
Your explanation makes sense. MAP2 is considered to be a marker for more mature neurons. I'm not completely sure to why you're using WB to quantify this though. Shouldn't you have to assess these markers by ICC and normalise them to total number of cells?
You can use nestin or musashi to distinguish the neural progenitor cells, and use NEUN for neurons if you haven't tried that yet. If your progenitors have a regional specificity, you could try some nuclear markers like PAX6, FOXG1, DMRTA2, OLIG2... depending on which brain region you're working with
Thank you so much. I already did ICC and have positive data. I want to have more evidences, so I tried Western blot. I am also thinking about NEUN. Your opinion has given me a clarity. Thank you once again :)
Sravan Goparaju Thank you so much for your clear explanation. That was helpful.
Daniel Cabezas de la Fuente Thank you for the clarification. What exactly does Foxg1 stain? And can you tell me about Nkx2.2 and Sox11?? Thank you in advance.
the markers above are for region-specific neurons, which introduces additional unneeded complexity to this experiment. If the question is whether the drug induces/promotes neuronal differentiation from rat NSCs, then use the simple and robust markers such as rat Nestin (developed by Ron McKay), rat-401 clone, available from DSHB, vs Map2, neuronal class III b-Tubuli (TUJ1 antibody), doublecortin, doublecortin-like kinase, Gap43. Describe changing ratios of Nestin/DCX, Nestin/Tuj1, Nestin/Map2 etc. If you are keeping cultures for a longer time then it makes sense to use markers of mature neurons e.g., NeuN, maybe even neurotransmitter mrkers, synaptophysin, vs. Nestin. Nestin is a great marker for this experiment. As NPCs mature then change from Nestin+ to Tuj1+, MAP2+ , also GFAP+ (at later passages you get a lot of astros). E.g, see our earlier papers PMID: 19609935 or even PMID: 16806174)
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