Immunocytochemistry for GluR1 Subunit of AMPA Receptors in Hippocampal Neurons

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Anatomy and Neurobiology
University of California Irvine
Post-doctoral researcher

Company:

Millipore

Product Name:

Rabbit anti-GluR1

Catalog Number:

AB1504

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. Glutamate receptors play an important role in learning and memory and rapidly regulate spine dynamics. 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

I used this antibody to co-localize AMPA receptors and CRH receptors to the same dendritic spines. I performed a serial dilution with 1:250, 1:500, 1:1000, and 1:2000 conditions. The dilutions 1:1000-1:2000 looked very punctate, and I chose 1:1000 to hold up to triple label confocal imaging.

Additional Notes

None

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Summary

The Good

The antibody produces a punctate immunocytochemistry consistent with proteins found in dendritic spines. Quantification of GluR1+ spines from this immunocytochemistry is consistent with the literature.

The Bad

Higher concentrations produce high signal in the soma.

The Bottom Line

The antibody produces a punctate immunocytochemistry consistent with proteins found in dendritic spines. Quantification of GluR1+ spines from this immunocytochemistry is consistent with the literature.

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