How to harvest RGCs from rat retina for protein analysis such as western?

QIN

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Nov 16, 2015
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#1
I have tried to do analysis of the change of some protein level in the retina ganglion cell,some scientists told me that I could use cryosection to get the ganglion cell layer then used for western blot. However, it is difficult to get very pure ganglion cell population. Would anybody has better suggestions?

Thank you and have a good day.
 

frasermoss

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Nov 16, 2015
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#2
This paper has an immunoselection protocol to make a 99% pure RGC prep

Neuron. 1988 Nov;1(9):791-803.Click here to read

Immunological, morphological, and electrophysiological variation among retinal ganglion cells purified by panning.

Barres BA, Silverstein BE, Corey DP, Chun LL.

Program in Neuroscience, Harvard Medical School, Boston, Massachusetts 02115.

Two different monoclonal antibodies to the Thy-1 antigen, T11D7 and 2G12, were used to purify and characterize retinal ganglion cells from postnatal rat retina. Although Thy-1 has been reported to be a specific marker for ganglion cells in retina, retinal cell suspensions contained several other types of Thy-1-positive cells as well. Nevertheless, a simple two-step "panning" procedure allowed isolation of ganglion cells to nearly 100% purity. We found that postnatal ganglion cells differed in antigenic, morphological, and intrinsic electrophysiological characteristics, and that these properties were correlated with one another. Minor variations of this panning protocol should allow rapid, high yield purification to homogeneity of many other neuronal and glial cell types.

PMID: 2908449
 

alissaw

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Nov 16, 2015
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#3
I'm a grad student in Ben Barres' lab (the first author on the neuron paper that frasermoss mentioned). We isolate and culture RGCs all the time and are happy to send out our current protocols. Ben's email is barres at stanford dot edu. It's a very involved prep, (takes about 6 hrs once you get it down) but you get a pure cell population out of it. Good luck!
 

TheFFM

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Nov 16, 2015
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#4
Do you have a write up of that protocol that you could post in the Protocols section here



http://www.scientistsolutions.com/a32-protocolslist-neuroscience.aspx



for everyone to read?





Many Thanks