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Author Topic: Anti-p53 antibody protocoll for fresh frozen brain  (Read 7200 times)

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Offline voland

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Anti-p53 antibody protocoll for fresh frozen brain
« on: July 14, 2008, 01:41:44 PM »
Hello,

I am trying to stain fresh frozen brain tissue with p53 antibodies. I have tried 3 antibodies but no staining both with immunohistochemistry and immunofluorescence. I use Triton X-100. As seen in other papers one should see staining even in normal cells. Is there any "good antibody" that works on brain fresh frozen tissue or any "tricky" protocoll step that I have missed. I use spleen tissue as positive control since I do not have any tumor tissue. I would be very! happy if I get help with this since this thing has been "bothering" me the last 6 months.

Thanks!

Anti-p53 antibody protocoll for fresh frozen brain
« on: July 14, 2008, 01:41:44 PM »

Offline richard03

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #1 on: July 14, 2008, 02:24:02 PM »
What antibodies have you tried? Can you post detailed procedure?

Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #2 on: July 14, 2008, 06:46:40 PM »
I have tried Pab 246 (sc-100) from santa cruz, G59-12 from BD Biosciences and a third one that I got from someone and dont know what exactly it is. I have stained with DAB and fluorescence. Briefly for DAB:
Day 1
I let the sections dry for 1 hour
PBSx3
Fixate in acetone -20C or formalin +4C for 10 minutes
H2O2 0,3% for 30 minutes
PBsx3
Block with PBS+1% Bovine serum albumin+ 0,3% Trito X-100
Aplly the primary antibody diluted in the above solution 1:50
Day 2
PBSx3
Secondary anti-mouse 1 hour diluted in PBS+BSA+Triton as above
PBSx3
ABC kit from Vector in PBS 1 hour
PBSx3
DAB kit from Vector 3 minutes
counterstain with HTX, dehydrate in alkohol and mount with DPX.

Any clues?

Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #3 on: July 14, 2008, 07:03:02 PM »
I am sorry I did not mention that it is rat brain tissue.
And also it is 3% H2O2.

Offline richard03

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #4 on: July 14, 2008, 08:38:01 PM »
The protocol looks fine. Can you re-embed the frozen tissue into paraffin and perform antigen retrieval procedure? The reason I am asking is that most of p53 antibodies I worked with did work well on paraffin sections with citrate buffer antigen retrieval.

Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #5 on: July 15, 2008, 04:09:12 AM »
Is there a protocoll for re-embeding. I have never worked with paraformaldehyde perfused tissue before. My tissue is perfused with cold PBS. I cannot find a single article that has stained for p53 on fresh frozen, all are on paraformaldehyde perfused. Theoretically, what is the advantage of re-embeding+antigen retrieval. Is it possible that the p53 is not expressed on my brain contused tissue (there are some articles that say it should be).

Offline richard03

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #6 on: July 15, 2008, 06:58:04 AM »
You can put the frozen brain block into cold formalin or paraformaldehyde and fix the tissue overnight. Then proceed with paraffin embedding procedure.

I have not seen articles using frozen sections for p53 staining so I assume this type of antibody may work better on paraffin sections. The purpose of re-embedding in paraffin is to do antigen retrieval as it is required for p53 staining, but you can not do antigen retrieval on frozen sections.


Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #7 on: July 15, 2008, 08:35:43 AM »
Excuse me that I ask again but I dont understand fully. Antigen retrieval is for retrieval of antigens that have been masked with parrafin/formaline. What is the point to mask the antigens first with parafin/formaldehyde and retrieve them later. Are they not "retrieved" from the beggining in the frozen sections? Maybe the articles for p53 do parrafin perfusion only for tissue morphology?
 

Offline richard03

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #8 on: July 15, 2008, 10:07:40 AM »
It may not be just "mask/unmask" issue. It may also involved antigen preservation or other unknown issues. Anyway, the importance is that most reports/articles used paraffin sections successfully.

Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #9 on: July 15, 2008, 10:11:42 AM »
But then is the p-53 antigen not already destroyed since I did not perfuse it with parrafin from the beggining?

Offline excalibur

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #10 on: July 15, 2008, 10:17:38 AM »
Processing to paraffin will dissolve out the lipids present in the tissue. This may help with some antibodies.

Are you getting any staining? What species is your tissue? I assume you are using a mouse primary antibody, because you are using a mouse secondary.

Have you tried going right into acetone or formalin after drying the sections, rather than rinsing in PBS before fixation?
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
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Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #11 on: July 15, 2008, 10:32:33 AM »
It is rat brain with traumatic brain injury. According to some reports there should be some staining in naive animals as well. I do not get any staining. A positive control for another antibody ED1 works, meaning that there were no problems with the protocoll. Maybe the antibodies do not work (they should be working on rat according to other reports). The problem is that I do not have tumor tissue to have as positive control. Should I buy a polyclonal? I have not tried to fix directly in formalin or aceton. I could try that. I have not done parrafin embedding and antigen retrieval before and I am a bit afraid that it is going to take a while until I get this new (for me) method to work but if you say that this may work I should do that?

Offline excalibur

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #12 on: July 15, 2008, 10:53:18 AM »
Also, the Santa Cruz antibody pab 246 does not state that it is for IHC.
This  is a list of their P53s: http://www.scbt.com/table-p53.html
Always look for an antibody that has IHC in its recommended uses list.

Although I have had to use some antibodies where IHC was not stated and had success.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Offline voland

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Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #13 on: July 15, 2008, 06:50:03 PM »
Yes, that is because I found an article (one of a few) that have stained with IHC on rat brain tissue and it was with PaB246, Anyway I have ordered a polyclonal antibody from Neomarkers, maybe I have more chance to see something with it, I hope it is not going to be unspecific staining.

Re: Anti-p53 antibody protocoll for fresh frozen brain
« Reply #13 on: July 15, 2008, 06:50:03 PM »