Hi Peter-

Since you are looking at receptor trafficking in cultured cells, I assume you are trying to differentiate surface NR2A/B from total NR2A/B with some kind of sequential labeling protocol.

Using an N terminal antibody to detect surface proteins prior to permeabilization with Triton-X , Tween, Saponin, etc will allow you to tag only those proteins considered to be "functioning". Subsequently, permeabilization after incubation with the N-terminal antibody will expose intracellular epitopes. Depending on how your receptor is modified post-translationally, you may be able to identify an internal or C-terminal epitope not present in the surface-bound, functional protein. Alternatively, if you can find two N-terminal antibodies, raised in different hosts, that recognize the same epitope, you can use two secondaries with different fluorophores to differentiate protein localization. I use a variation of the protocol outlined in the following paper, which is a good starting point:

http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.220.html

This protocol can be modified to study receptor turnover at various time points by incubating live cells with N-term antibodies prior to fixation.

Signal intensity has been a problem for me with NMDA subunit ICC in the past. Quenching PFA with a weak glycine solution prior to block helped a bit. Playing around with block solution concentrations also helped.

Good luck!

Dear peter we work on Hippocampus NMDA receptors rat and our protocols was use PFA 1% for 7 days also we use dRabbit polyclonal anti-NMDAR2B Millipore 06-600, 1:200 and Rabbit polyclonal anti-NMDAR1 Abcam AB28669, 1:200 and secondary antibay was DAB.

Hi Peter-

Since you are looking at receptor trafficking in cultured cells, I assume you are trying to differentiate surface NR2A/B from total NR2A/B with some kind of sequential labeling protocol.

Using an N terminal antibody to detect surface proteins prior to permeabilization with Triton-X , Tween, Saponin, etc will allow you to tag only those proteins considered to be "functioning". Subsequently, permeabilization after incubation with the N-terminal antibody will expose intracellular epitopes. Depending on how your receptor is modified post-translationally, you may be able to identify an internal or C-terminal epitope not present in the surface-bound, functional protein. Alternatively, if you can find two N-terminal antibodies, raised in different hosts, that recognize the same epitope, you can use two secondaries with different fluorophores to differentiate protein localization. I use a variation of the protocol outlined in the following paper, which is a good starting point:

http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.220.html

This protocol can be modified to study receptor turnover at various time points by incubating live cells with N-term antibodies prior to fixation.

Signal intensity has been a problem for me with NMDA subunit ICC in the past. Quenching PFA with a weak glycine solution prior to block helped a bit. Playing around with block solution concentrations also helped.

Good luck!

Try incubation the cell sides in cold room (4C) for 2-3 days after add on the 1st. antibody. This how we did on cultured cells.

Please see a list of antibodies and the conditions in the paper and supp data. A lot in vivo, but I hope some help for another alternatives.

B.

Thank you all for sharing your experiences with NMDAR staining! Especially the nature protocol paper helped me a lot. I read more on cell culture-treatment prior to NMDAR staining and discovered that most people fixate neurons in -20 degrees methanol for 10 min.

As you say Grant, I’m interested in studying the cellular expression of NMDAR and be able to distinguish between surface vs. non-surface bound and furthermore synaptic vs. extra-synaptic receptors. But first of all I wish to succeed staining at all…

I will try methanol fixation and experiment with 4% PFA on ice to see how it affects the immunostainings. I will let you know when i'm done!

Peter

Hi Peter,

I followed:

http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.220.html

Crucially, I avoid fixing with PFA. Instead, fix with -20degC methanol. Fix cells on ice for 7 mins. Then wash with water, then PBS, and carry on immunostaining as normal ( block with 3% BSA in 0.1% triton for 1 h; make up antibodies in blocking buffer; primary o/n at 4degC; 3x 5 min wash in PBS; secondary for 2 h at RT; 3x 5 min wash in PBS) . The BD Ms GluN2B antibody works very well as does the Invitrogen Ms GluN1 antibody (this monoclonal is also available from lots of other suppliers).

Good luck,

Rene

Hi Peter, I'm curious, did methanol fixation and the mouse BD antibody work for you? I need to stain neuronal cultures for GluN2A and GluN2B and wonder which antibody to purchase.

 Best, Yvonne