Hi Betty,

at which day in vitro do you normally stain your cultures? At div 21 could be too early. Synaptogenesis and synapse maturation depends on the neuron density. Changes of this parameter may explain variable results. E16 is very early, implicating that synaptogenesis may be slower compared with cultures made at E17 or E18, which relates to the above.

I would not wash cells with PBS before fixation as this may cause dispersial of postsynaptic receptors due to lateral diffusion in the plasma membrane in response to cell stress. Btw, why don't you simultaneously incubate all first antibodies? This works well and safes time. I would not incubate first antibodies over night as bacteria may grow and blur your signal, except if you use NaN3. One hour at RT is sufficient. 

If you wish I could suggest other NR1 antibodies tomorrow which worked well in our hands. Also consider using NR2 antibodies.

Hope this helps.

Kind regards

Jochen 

Dear Jochen,

Thank you very, very much for your answer!! It helps me a lot!

I normally stain my cultures at  DIV 21 as I thought this would be a good time point. Members of other labs start to stain them at DIV14, so I thought with DIV 21, I would be on the safe side. Do you culture neurons from rat or mice? Do you grow them on an astrocyte layer?

Good point you mentioned with the density. This is a parameter that varies indeed. What would you say is a crucial density for neuronal culture without astrocytes?

I stained consecutively because of the extra- and intracellular antigenes.

Bacteria grow over night in the fridge? I thought it would be a problem if I would stain over 3 days or so. But this a step, like washing with PBS, I can change easily.

Sure, I am interested in other NR1 antibodies. As we are only investigating NR1 subunit, my focus lies on NR1.

Thank you very much!!!

Best, Betty

Dear Betty,

We do both rat and mouse. Plating density is 90,000 per cm2. We naturally have astrocytes in the cultures as we normally don't use AraC. Why don't you try different plating densities and determine the best time point for NR1 staining?

Regarding antibodies: NR1, Chemicon, AB5048P; NR2B, Chemicon, AB1557P. For reasons of curiosity and scientific interest I would also check NR2 expression even if this subunit is not your main topic.

Regarding bacteria in the fridge: Yes they grow, but much slower than at 37°C or RT... They are fluorescent and even a low number of bacteria may add to your NR1 signal.

Is the epitope on the NR1 subunit surface accessible on living neurons? If so, I would stain 5-10 minutes in culture medium in the incubator, wash 3 times with medium and then fix the cells. This will give you a true surface signal. FYI, please see my contribution to the discussion "Does anyone know if PFA fixation changes the antibody affinity/recognition for/of cell surface antigens?".

Cheers,

Jochen

Dear Jochen,

Thank you very much for your answer. BTW, we are sitting at the CCO at the Charité Campus Mitte. Not that far away from Buch ;-)

I also have some astrocytes in my cultures as I don't  use any DNA-Replication inhibitor. And sure, the older the culture gets the more astrocytes I have in the culture. I was just wondering as some people told me it is nice for stainings to have as less astrocytes as possible. But on the other hand, astrocytes' presence ensure a more physiological environment for neuronal growth.

BTW, I just treat coverslips with PLL and Collagen. I don't perform any heating or washing but heard from other groups doing so. Do you treat your coverslips?

The epitope is accessible in living neurons. You are totally right! I will try the next staining with fixation after antibody incubation. Afterwards, I can still stain for pre-synaptic markers.

Also, thanks for the link! Very informative!

Cheers, Betty

Dear Betty,

Great, just step by if you are at the MDC in Buch and we could discuss these things in a bit more detail.

Cheers,

Jochen

Hey everybody,

we did succesfully stain Neurons with anti-NR1 AB. They had nice clusters (see 1st picture) and were suitable for cluster quantification. But since several weeks, we can not reproduce our staining in neuronal culture (see 2nd picture). In transfected HEK-cells and in WB-analysis the AB works fine. We also tried 2 other anti-NR1 AB with the same result.

The neurons look healthy (see 3rd and 4th). The only thing we changed is the incubation of the cover slips with 10% PLL instead of undiluted.

We are now wondering what is going wrong? Or  are theses tiny "clusters" even more physiological? Do you have any suggestions?

Best wishes, Betty

Hi Betty,

10% PLL means Poly-L-Lysine? Via cell adhesion contacts neurons interact with the ground they are growing on. No idea whether 10% vs. undiluted makes a difference, but in any case I stick to the same neuron-culturing procedures while comparing different experimental conditions - don't forget that cell culture is an artificial system...

Cheers,

Jochen