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Author Topic: Antigen Retrieval problem 4%PFA perfused, acetone fixed  (Read 13761 times)

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Offline Jogi

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Antigen Retrieval problem 4%PFA perfused, acetone fixed
« on: January 28, 2008, 12:31:08 PM »
I am quite struggling with some slide here in the new lab - (for BrdU and Ki67 - which does not make it easier).
Anyway, the brain was perfused with 4%PFA, kept in PFA o.n. and afterwards kept at 4C in sucrose (unfortunately, quite long for about 2 months)

Thereafter, brain samples were frozen to -80C, sliced, air dried for several hours, acetone fixed for 15min. air dried, and kept till usage in the -80C.

So now I have the problem that all different antigen retrieval methods i used, failed more or less completely. 
what i used are the following:
HCL for 30min. as well as 2h @37C, afterwards boric acid buffer wash
Citrate Buffer retrieval:
CB, ph3 for 2h at 37C
CB, ph6 in the microwave (whole protocol, just 9min., tried to keep the balance between not overboiling and still keeping it at around 95C)
CB, ph6 in water bath for 1h at just 90C (could not achieve higher degrees)

for permeabilization i used 1%SDS as detergent, I am gonna change that probably to 0,25% Triton-X

any ideas? I am very grateful for any comments and suggestions. any information missing? can also post the whole protocol, but I am 100% sure it is the tissue processing, and AR. thx


Antigen Retrieval problem 4%PFA perfused, acetone fixed
« on: January 28, 2008, 12:31:08 PM »

Offline excalibur

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Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #1 on: January 30, 2008, 09:43:47 AM »
Antigen retrieval is not neccessary with frozen sections.

Your problem may be with underfixation and the tissue being stored for 2 months in sucrose.

What species are the brains?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline Jogi

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Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #2 on: January 30, 2008, 01:03:37 PM »
it is rat tissue;

would suggest further PFA treatment?

Offline excalibur

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Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #3 on: January 30, 2008, 01:25:46 PM »
Yes, overnight is not enough time to fix rat brain.

And it is better to freeze the tissue or cut sections as soon a possible. Don't leave in sucrose for months.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline CanuckPhD

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Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #4 on: January 31, 2008, 01:37:34 AM »
Overnight fixation of a perfused rat brain is long enough. I have had very good results and morphology from this. You just have to perfuse the rat with 50-80 mls of 4% PFA over 5-10 minutes, harvest the brain, remove the cerebellum and olfactory bulb if not needed and fix ON. In fact if you block the brain in slabs of 0.5 cm, a 3 hour fixation is good enough.

As for the sucrose, I have never left the brains longer then 4-5 weeks in 15% sucrose so can't comment on that.

My question is do you know for sure that you should be seeing proliferating cells in your brains? In normal and many pathological conditions these are rare events, so maybe they are just not there.

I agree do not use antigen retrieval in frozen tissue, though the HCl is likley necessary for the BrdU


Offline Jogi

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Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #5 on: January 31, 2008, 03:12:49 PM »
thx for the comments already!!!

regarding sucrose: Unfortunately when I came to the lab the brains were already in sucrose for about 2 months, but I think as long as nothing else is growing in there it should be ok!?

regarding brdu in the cells: rats were injected with BrdU for 7 days, so they should show something. The problem is I do not see any staining at all (no brown background from the dab or else, though ABC complex, and DAB are working fine) so there is something more fundamental wrong - ( for BrdU rat anti brdU from abcam (1:100) o.n. at 4C + Rabbit anti Rat for 30min. (1:500) is used. ) and  - now it does not really fit anymore in this Antigen Retrieval forum  ;D

I am now trying no acetone step, no antigen retrieval just HCL or detergent!
and also some more common and easier antigens just like GFAP to test the protocol in general, though if the primary is working I am sure there is at least nothing fundamental wrong.


Re: Antigen Retrieval problem 4%PFA perfused, acetone fixed
« Reply #5 on: January 31, 2008, 03:12:49 PM »