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Author Topic: reusing antibodies and contamination  (Read 5689 times)

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Offline frenchkl

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reusing antibodies and contamination
« on: September 28, 2005, 09:11:17 PM »
I would like to know if anyone has experience reusing antibodies when doing immunohistochemistry?  I expect that success with reusing antibodies depends on the antibody, but I am particularly concerned about contamination.  In my protocol, I incubate for 3 nights at 4oC, and I use normal goat serum in my primary antibody solution.  Typically, I see some debris when I remove my sections from the antibody incubation solution, so I am not inclined to reuse this solution.  On the other hand, our lab is trying  to conserve antibody.  Does anyone have opinions on this or information that might help me decide?

Thanks!
risten French
MUSC

reusing antibodies and contamination
« on: September 28, 2005, 09:11:17 PM »

Offline excalibur

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reusing antibodies and contamination
« Reply #1 on: September 29, 2005, 01:54:54 PM »
I have never reused antibodies.

The debris you are seeing is probably from incubating 3 nights.
This seems excessively long and the debris may be bacterial growth. One night incubation is usually sufficient.

How thick are your sections?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline frenchkl

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reusing antibodies and contamination
« Reply #2 on: September 29, 2005, 03:17:24 PM »
We often incubate at least 48 hours or over the weekend, so this doesn't seem excessive to me.  However, I do think that the debris is suspicious.

My sections are 45 microns.
risten French
MUSC

Offline excalibur

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reusing antibodies and contamination
« Reply #3 on: September 29, 2005, 03:24:09 PM »
OK, I thought the long incubation time would only be done for thick sections. You might let one of the slides dry flat so you don't lose fluid, fix with methanol, and do a Gram stain for bacterial growth though.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline ImmunoNYC

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reusing antibodies and contamination
« Reply #4 on: September 30, 2005, 12:06:07 AM »
I never reuse antibodies after application to tissue and shun the idea although someone in my lab does it much to my dismay. I shudder to think of the variation in staining caused by doing this.

Three nights also seems excessive to me as I do one hour at RT for 99% of my stains. You might get better results by going shorter even to one hour. But then again, I am of the mindset that if it ain't broke, don't fix it.

Offline richard03

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reusing antibodies and contamination
« Reply #5 on: September 30, 2005, 07:17:58 AM »
I am on the same side as Paula and MaximinaNYC that three night incubation seems excessively long. I have done mouse brain paraffin sections at 50 um thick for 2 hours TH staining and it worked just fine. So one night is sufficient.

I have reused antibodies many years ago due to budget issue and some antibodies in short supply. Now I never reuse antibodies anymore.

Richard

reusing antibodies and contamination
« Reply #5 on: September 30, 2005, 07:17:58 AM »