ICC for CRH Receptor Type 1

January 07, 2014

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Anatomy and Neurobiology
University of California Irvine
Post-doctoral researcher

Company:

Everest Biotech

Product Name:

Goat anti-CRH receptor type 1 (N-terminus extracellular epitope)

Catalog Number:

EB08035

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

1:2000

Blocking Agent

3% BSA + 0.1% Triton-X100

Secondary Incubation

1:400

Tertiary Incubation

None

Detection

Fluorescence

Results Summary

I fixed cultured dissociated hippocampal neurons for 12 minutes with 4% PFA in PBS on ice. I performed a serial dilution of the primary antibody and found 1:2000 produced a punctate immunofluorescence in the soma and dendrites of neurons that is indicative of proteins found in dendriitc spines. A previous study tried several antibodies in receptor WT and receptor KO tissue samples (see Refojo et al., 2011) and found no adequate antibody to detect CRH receptor type 1. This receptor has been localized to dendritic spines using EM (see Chen et al., 2004) and immunofluorescence detection in Thy-1 YFP expressing mouse tissue samples (see Chen et al., 2013). While a more reliable antibody is necessary to study CRH receptor type 1 in neurons, this is a decent antibody to try in your sample.

Additional Notes

None

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Summary

The Good

Antibody produces a punctate stain that co-localizes with PSD95 and to the heads of dendritic spines in rat cultured hippocampal neurons and Thy-1 expressing mouse tissue

The Bad

Reliability of commercially available antibodies to CRH receptor type 1 has been questionable. Several groups have published several different results.

The Bottom Line

While a more reliable antibody is necessary to study CRH receptor type 1 in neurons, this is a decent antibody to try in your sample.

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