Immunocytochemistry for NR2A Subunit of NMDA Receptors in Hippocampal Neurons

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Anatomy and Neurobiology
University of California Irvine
Post-doctoral researcher

Company:

Thermo Fisher

Product Name:

Rabbit anti-NR2A

Catalog Number:

PA3-103 (discontinued)

Stress in vivo, and CRH application in vitro, cause dendritic spine loss in hippocampal pyramidal neurons. This loss of spines, and subsequent synapses, is likely the underlying mechanism through which severe stress disrupts learning and memory. CRH, in combination with neuronal activity and NMDA receptor activation, recruits the actin destabilizing protein, calpain, to break down the actin cytoskeleton resulting in spine loss. Identifying the downstream signaling from CRH receptors could reveal potential therapeutic targets for diseases such as post-traumatic stress disorder (PTSD), anxiety, and depression.

Experimental Design and Results Summary

Applications

Immunofluorescence

Sample

Cultured hippocampal neurons

Primary Incubation

1:1000

Blocking Agent

3% BSA + 0.1% Triton-X100 in PBS

Secondary Incubation

1:400

Tertiary Incubation

None

Detection

Fluorescence

Results Summary

This antibody was used to localize NMDA receptors and CRH receptors to the same dendritic spines. Neurons were fixed with 4% PFA in PBS pH = 7.4 on ice for 12 minutes before immunocytochemistry. Several serial dilutions were performed with 1:1000 being the best for confocal imaging. This antibody produces a punctate state consistent with dendritic proteins. Quantification of the number of NR2A+ dendritic spines was consistent with the literature.

Additional Notes

None.

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Summary

The Good

This antibody produces a punctate state consistent with dendritic proteins. Quantification of the number of NR2A+ dendritic spines was consistent with the literature.

The Bad

None.

The Bottom Line

This antibody is excellent for localization of NMDA receptors in hippocampal dendritic spines. I tried several antibodies against the obligate NR1 subunit and none of those antibodies produced a reliable immunocytochemsitry.

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