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Author Topic: problem with nestin immunohistostaining  (Read 3631 times)

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Offline zhiyongchen

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problem with nestin immunohistostaining
« on: September 06, 2005, 03:59:33 PM »
I got a great immunocytostaining with Chemicon Nestin monoclone antibody at 1:200 dilution. However, when I tried the same antibody on immunohistochemitry, I did not get a staining even at 1:20 dilution. I perfused the mouse brain with 4% PFA, postfixed in 4% PFA overnight, treated with 30% sucrose and then cut frozen sections and did staining with float sections. Any suggestions? Thanks a lot!

problem with nestin immunohistostaining
« on: September 06, 2005, 03:59:33 PM »

Offline richard03

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problem with nestin immunohistostaining
« Reply #1 on: September 06, 2005, 05:04:01 PM »
Did you use the same fixative (4% PFA) for immuncytostain?

Offline zhiyongchen

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problem with nestin immunohistostaining
« Reply #2 on: September 07, 2005, 07:58:04 AM »
Yes, I did. Everything was same.

Offline richard03

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problem with nestin immunohistostaining
« Reply #3 on: September 07, 2005, 08:06:12 AM »
Maybe longer incubation will work. How long did you incubate your primary? You may also add some Triton X-100 in primary incubation buffer and it will help the penetration of antibodies.

Richard

Offline zhiyongchen

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problem with nestin immunohistostaining
« Reply #4 on: September 07, 2005, 08:28:06 AM »
Dear Richard,
Thank you for your advice. However, I incubated my sections with first Ab (nestin) for 3 days and I did include 0.01% triton in my buffer for every step.

Offline richard03

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problem with nestin immunohistostaining
« Reply #5 on: September 07, 2005, 09:18:02 AM »
My next suggestion is to use antigen retrieval method below and it may helps:

http://www.ihcworld.com/_protocols/epitope_retrieval/free_floating_sections.htm

Or dilute your primary antibody more to 1:1000 or 1:2000. There were several antibodies I used before and they were working with high dilution but not low dilution. I don't know why it should work that way but it works sometime.

Richard

problem with nestin immunohistostaining
« Reply #5 on: September 07, 2005, 09:18:02 AM »