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Author Topic: IF on formalin-fixed paraffin-embedded tissues  (Read 17970 times)

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Offline annie1

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IF on formalin-fixed paraffin-embedded tissues
« on: October 14, 2004, 12:29:32 PM »
Can anybody recommend a good protocol for performing immunofluorescence on FFPE human liver and colon sections? I am specifically interested in labeling intercellular junctional proteins. Thanks for any advice!

IF on formalin-fixed paraffin-embedded tissues
« on: October 14, 2004, 12:29:32 PM »

Offline brian

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IF on formalin-fixed paraffin-embedded tissues
« Reply #1 on: October 14, 2004, 01:06:10 PM »
Do you need general protocol or a protocol for particular antibody?

Brian

Offline annie1

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IF on formalin-fixed paraffin-embedded tissues
« Reply #2 on: October 15, 2004, 06:02:31 AM »
Just a general protocol - I have only ever used frozen sections for IF, so I'm not sure about the best way to proceed after de-paraffinization.  Thanks, Annie

Offline brian

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IF on formalin-fixed paraffin-embedded tissues
« Reply #3 on: October 15, 2004, 02:38:42 PM »
In general, IF on frozen sections is same as IF on paraffin sections except that paraffin sections often required antigen retrieval step.

Check the following link for a general IF protocol.

http://www.ihcworld.com/_protocols/general_IHC/immunofl.htm

Good luck.

Brian

Offline annie1

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IF on formalin-fixed paraffin-embedded tissues
« Reply #4 on: October 16, 2004, 06:34:37 AM »
I have heard that after de-paraffinization some people just fix in alcohol and detergent-permeabilize as if for normal IF on frozen sections - could detergent-permeabilization be as efficient as antigen-retrieval? I had assumed a majority of antibodies would require antigen retrieval.

Offline richard03

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IF on formalin-fixed paraffin-embedded tissues
« Reply #5 on: October 16, 2004, 08:51:02 AM »
Detergent-permeabilization will open cell membrane and is to increase antibody penetration into the tissue and cell.

Antigen-retrieval will break cross link formed during formalin fixation and paraffin embedding and is to enhance antibody binding onto antigens/epitopes of the tissue and cell.

Fresh frozen sections are usually fixed in acetone or alcohol so is no need to do antigen retrieval. Only detergent-permeabilization is sufficient.

Paraffin sections are usually fixed in formalin so both detergent-permeabilization and antigen-retrieval will be needed to uncover antigens/eiptopes. Since paraffin sections are usually very thin (3-6um), most of antigens/epitopes are already exposed thus detergent-permeabilization may not be necessary.

The following is a general procedure for FFPE sections:

1) Deparaffinize sections in xylene, 2x5min.  
2) Hydrate with 100% ethanol, 2x3min.
3) Hydrate with 95% ethanol, 1min.
4) Rinse in distilled water.
5) Antigen retrieval
6) Rinse in PBS
6) Normal serum blocking (containing detergent-permeabilization such as Triton X-100)
7) Primary antibody
8) Standard procedure...

Hope this helps.

Richard

Offline annie1

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IF on formalin-fixed paraffin-embedded tissues
« Reply #6 on: November 02, 2004, 07:45:51 AM »
Thanks to both Richard and Brian - very helpful!

Offline colin23

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did this work?
« Reply #7 on: February 18, 2005, 04:41:24 PM »
Hi all,
Just wondering if this worked or not? I've tried what richard has suggested but get tons of autofluorescence.

I'm working with formalin fixed paraffin embedded mouse colon. if that helps at all...

Cheers
Colin

Offline annie1

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IF on formalin-fixed paraffin-embedded tissues
« Reply #8 on: February 21, 2005, 04:46:25 AM »
I tried almost everything to reduce autofluorescence in FFPE liver tissues, and mostly saw almost no benefit. The only thing that worked somewhat was copper sulfate in an acidic buffer. I found it in this reference:
J Histochem Cytochem. 1999 Jun;47(6):719-30.
Good luck!

Offline richard03

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IF on formalin-fixed paraffin-embedded tissues
« Reply #9 on: February 21, 2005, 12:51:16 PM »
Once the tissue has autofluorescence, you can "REDUCE" it but almost impossible to "erase" it completely. My suggestion is if the tissue had strong autofluorescence, the best way is to switch to enzyme detection methods.

Richard

Offline greiner

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IF on par. sections without avidin-biotin system?
« Reply #10 on: May 10, 2005, 06:23:37 AM »
Hi, everybody,

for the last 4 months I am doing IF on fibroblasts and it works just fine. Now I am trying to do IF on paraffin sections using the same antibodies as for IF on cells. It doesn't work. I did antigen retrieval with citrate buffer, pH6 as recommended by ihcworld. I used microwave and boiled samples for 10, 20 and 40 minutes with adding water in between, I tested different dilutions of primary Ab (1:20 to 1:100). As I read on these pages avidin-biotin detection system is common step in protocols. Is it possible to do IF on paraffin sections without this signal amplification? The antibodies I used are anti-C23 (Santa Cruz #8031) and anti Cdk2 (Santa Cruz #M2), both of which work on IHC.

I am grateful for any advice! :)

IF on par. sections without avidin-biotin system?
« Reply #10 on: May 10, 2005, 06:23:37 AM »