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Post subject: Immunofluorescence - GMA embedded human tissue sections
Could anyone suggest me a good antigen retreival step to perform immunofluorescence on GMA embedded human tissue sections Please.
Thanks in advance.
Thu Feb 23, 2012 11:13 am
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1872
Post subject:
Mount sections on charged slides...place in 60C oven for at least 60 mins.
Heat your antigen retrieval solution to 90C in a water bath, for eg.
Then add your sections.
They shouldn't come off but, this is not a guarantee.
Heat them at a higher temp, and mine fall off.
Make sure that you give incubations times between 20-60mins.
You MUST include a reliable irrelevant primary Ab control that you know MUST be positive ( eg: I used anti GFAP when doing GMA brain sections).
If this does not work....forget it and do Pwax sections.
Unlike some others, I have not been able to get decent IF nor IHC on GMA-embedded sections.
Imho, the Abs cannot penetrate the polymerised resin with reliable, consistent success.
NB: why do you wish to use GMA-embedded sections for IF?
It is difficult, imho.
Curious Carl
Thu Feb 23, 2012 6:12 pm
surya
Rank: Member
Joined: Feb 23, 2012
Posts: 3
Post subject:
Thanks carl. There are many embedded samples in GMA already and they are all precious. As part of my project, If I could make this work, I will be able to use these samples without waiting for ages for the new one's.
Actually, I was planning to do PIER rather than HIER. I read somewhere that enzyme treatment is good for these resin section. Have you tried pronase or trypsin before?
thanks a lot again for your reply
Fri Feb 24, 2012 4:00 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1872
Post subject: Ta for info, surya
I have tried several proteases and also attempted to etch the GMA.Nothing worked for me...
However, it must be worth a try for you .
The main trouble is that GMA polymerises into a very tight structure.
Sure, they say to add less hardener/polymeriser but, I ended up with blocks so sticky the sections just "mushed" up.
However, a established/published method has been reprinted by Abcam I noted:
http://www.abcam.com/ps/pdf/protocols/gma.pdf
I did try this with no consistent success, even with very good primary Abs ( eg: after applying Dako's Rb anti GFAP only part of the specimen would be moderately positive).
Anyway, if your specimens are fixed in Formalin...forget it.
Why did you commit to GMA without having established a working method, , may I ask?
Even more curiously,
Carl
Fri Feb 24, 2012 4:51 pm
surya
Rank: Member
Joined: Feb 23, 2012
Posts: 3
Post subject:
As I was mentioning before, these samples were embedded 3 or 4 years back. I recently started my PhD and my supervisors thought that it would be good to use these samples and I am trying to optimise the protocol. They have done some immunohistochemistry before but not immunofluorescence.
Fri Feb 24, 2012 5:03 pm
Carl
Rank: Member
Joined: Oct 11, 2004
Posts: 1872
Post subject: Ah...
If IHC has previously worked, using the same prep. method must also work for IF.
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