PSD-95 known as synapse associated protein, is one of a family of membrane-associated proteins found in the postsynaptic density in forebrain neurons and certain presynaptic structures in the cerebellum. In this case, it only can be stained in the synapse of certain neurons. I guess mouse neuron which you used from the primary culture. I stained this kind of neurons and we can get very nice staining when cultures are more the 3 weeks. However, stem-cell derived neurons is quiet different with primary neurons because stem-cell derived neurons have much less synapses even you can see long and dense processes. So this is the reason your staining is not good in stem-cell derived neurons. 

What would you suggest as a plating density for the neurons in order to see a good amount of synapses?

I would agree with Dongliang that maybe the problem is that there are no functional synapses present in your cultures. Did you ever check that for example with multi-electrode arrays? If you look at the density of neurons between the primary and your stem cell-derived cultures as well as the morphology of the neurons, I think that it would not be too far fetched to first make sure that there should be synaptic PSD-95 present. Did you ever check the morphology of the dendritic spines (the place where the PSD-95 should be located)? If there are a lot of mushroom-shaped, mature spines, then there also should be PSD-95 be present...

Hi Mandy, another reasen why anibodies are not so working like you expect it is the reactivity. Make shure that your ab is working on human tissue or cells.  A suitable ab might be the PSD95 from  Cell Signaling #2507. An other thing is the concentration of your ab. May be you need a higher concentration of your ab when you are working with human tissue. If you do not like to increase the concentration of your ab such much  try to detect the ab binding with biotinylated secondary ab and streptavidine CY3 or what colour you like. Strepavidin-immuno enhances the staining result.

Good luck ! Ute

Hi Christian, sorry I didn't answer for so long, I didn't get a message that anyone had responded other than on the first day. So the Ngn2 neurons don't have dendritic spines per se, but they do have functional synapses because they fire in MEA. I also checked the PSD-95 mRNA levels at that age via single-cell seq and it's definitely present. I did a WB to check, and there is PSD-95 signal, although not very strong. With my best PSD-95 antibody, there is faint staining in the soma at times, but mostly it's just completely blank. So far I have been able to get the staining to work only once, in a select few wells, and with very old neurons (~3mo). I think it is just unlikely that PSD-95 is localized into distinct synapses in young Ngn2-derived neurons. Do you have any other post-synaptic marker suggestions I might be able to try? I've been thinking about using Homer1. 

Hi Ute, I definitely tried multiple Ab concentrations ranging from 1:100 all the way down to 1:2k. I do like your idea of using the biotin-streptavidin to increase the signal. Thank you!

I was able to get this to work. Going based off of RNA-seq data alone, I thought PSD-95 would show up at day19 because that's when the mRNA is expressed. However, it didn't end up showing up until after 3 months of growing the neurons, but it does look beautiful in my IF! 

Hello Mandy, 

Do you add astrocytes to your NGN2 human neurons?

Best regards,

Ashutosh

Hi Ashutosh,

Yes, after a 4 days I believe they are plated on mouse glia.

Mandy

Mandy Herring Hi Mandy! This was a long time ago but by any chance do you remember which one of your antibodies worked the best on 3 month neurons?

Hi! I work with hiPSC derived human neurons differentiated with StemCell Forebrain Differentiation and Maturation kits and in my case neurons start to consistently express PSD-95 around D28 of neuronal maturation.

Thanks for getting back to me. I see, I am working with hiPSC derived NGN2 neurons and have had no luck detecting PSD95 by day 21 although I am culturing them on top of the astrocyte. I might need to culture them longer then! The antibody I use is from Thermo (MA1-046). do you suggest any other antibodies?