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Author Topic: staining glutamatergic neurons in culture  (Read 7466 times)

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Offline immunologist

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staining glutamatergic neurons in culture
« on: November 19, 2009, 04:36:58 PM »
Please let me know the protocol for staining glutamatergic neurons in culture. Is the folloing protocol OK? IS there any aoher antibody other than vGlut1 and vGlut2?

Fix with 4%PFA (Room Temperature)

Wash with PBS

Block with blocking buffer (10%FCS in PBS)

Add vGlut2 in blocking buffer (incubate overnight)

wash with PBS

Add secondary antibody (Room temperature, in dark)

wash and mount.

staining glutamatergic neurons in culture
« on: November 19, 2009, 04:36:58 PM »

Offline richard03

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Re: staining glutamatergic neurons in culture
« Reply #1 on: November 21, 2009, 02:00:14 PM »
The protocol looks fine. Just remember to add Triton X-100 (0.5%) in blocking solution as well as primary and secondary antibody incubation.

Offline immunologist

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Re: staining glutamatergic neurons in culture
« Reply #2 on: November 23, 2009, 03:34:06 PM »
Thanks for the reply. I added 0.1 % triton in the blocking buffer, but not in the antibody solution.

I did not get staining for vGlut2, although Westernblot data done in parallel shows signal.

Any further suggestions are highly appreciated.

Thanks.

Offline richard03

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Re: staining glutamatergic neurons in culture
« Reply #3 on: November 23, 2009, 10:39:33 PM »
You may want to increase Triton conc. to 0.5% in blocking and antibody dilution buffer, at least in primary antibody dilution buffer. If this is still not working, you may consider other alternate fixation methods.

http://www.ihcworld.com/_protocols/general_ICC/fixation.htm

Offline immunologist

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Re: staining glutamatergic neurons in culture
« Reply #4 on: November 28, 2009, 02:44:31 AM »
Thanks again for the suggestion.

 Now, with triton in the blocking buffer and antibody solution, vGlut staining works.

But, simultaneous staining for MAP2 also looks dotted. When I do not use triton in primary and secondary antibody solution, MAP2 staining works.

I used 0.5%triton in blocking buffer and primary and secondary antibody solution.

Primary antibody was incubated overninght (4 degree centigrade) and secondary antibody was incubated for 3 hours.

Any suggestions are highly appreciated.

Thanks.


Offline richard03

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Re: staining glutamatergic neurons in culture
« Reply #5 on: December 04, 2009, 09:29:10 PM »
You may reduced Triton concentration to 0.3% to balance between vGlut and MAP2.

Re: staining glutamatergic neurons in culture
« Reply #5 on: December 04, 2009, 09:29:10 PM »