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Author Topic: de and restaining of histology slides  (Read 6879 times)

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Zara

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de and restaining of histology slides
« on: February 23, 2010, 07:21:58 AM »
Hi,

I have some tissue sections (Paraffin embedded fixed with PFA).
They are stained with first antibody and second antibody (fluorescence staining Cy3).
I used them for confocal laser scanning microscopy.
Now, they have been bleached out.
Is there any possibility to "rescue" these slices, to demount them (remove the vectashield, the coverslip and the antibody and the dapi) and to restain them again.

help and recommandations would be great  :-)

Thanks a lot
zara

de and restaining of histology slides
« on: February 23, 2010, 07:21:58 AM »

Offline folive

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Re: de and restaining of histology slides
« Reply #1 on: October 21, 2010, 09:35:56 PM »
I have the same issue.....DyLight 488 fluorophore has faded due to age of sections, but I want to save my precious tissue samples.  I used FluoroMount G as the mounting media, which is aqueous and should allow for "dissolving" the media to remove the coverlip (carefully, after removing the coverslip sealant). The manufacturer suggested I try remounting the sections in Prolong Gold and that should revive the signal. Would this work? Alternatively, I could re-stain, but wouldn't all the antigen sites be saturated with the primary or secondary antibodies? I suppose I could try both and see what happens. If anyone has any advice, I'm all ears. Thank you!  :)

Offline Cardio

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Re: de and restaining of histology slides
« Reply #2 on: October 22, 2010, 11:20:17 AM »
I have the same issue.....DyLight 488 fluorophore has faded due to age of sections, but I want to save my precious tissue samples.  I used FluoroMount G as the mounting media, which is aqueous and should allow for "dissolving" the media to remove the coverlip (carefully, after removing the coverslip sealant). The manufacturer suggested I try remounting the sections in Prolong Gold and that should revive the signal. Would this work? Alternatively, I could re-stain, but wouldn't all the antigen sites be saturated with the primary or secondary antibodies? I suppose I could try both and see what happens. If anyone has any advice, I'm all ears. Thank you!  :)

I believe you are correct. Unless you remove the antibody your epitope will be bound by your primary ab. I have never tried this so take it for what it is worth but if you used a different primary that bound to a different epitope site that would circumvent the problem. Personally I would generate more slides before doing such a thing.

Alternatively you could just do a DAB reaction with your samples but I am sure you thought about that before doing IF.
« Last Edit: October 22, 2010, 11:26:22 AM by Cardio »

Offline MT Scientist

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Re: de and restaining of histology slides
« Reply #3 on: October 22, 2010, 04:09:38 PM »
Alternatively, I could re-stain, but wouldn't all the antigen sites be saturated with the primary or secondary antibodies? I suppose I could try both and see what happens. If anyone has any advice, I'm all ears. Thank you!  :)

You may wish to look into methods of bound antibody removal by way of pH or Salt concentration.  These methods exist for Western blots and may very well exist for immunostains.

Offline excalibur

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Re: de and restaining of histology slides
« Reply #4 on: October 24, 2010, 11:17:01 AM »
For long term storage, you could use a chromagenic substrate such as DAB or AEC.
Paula Keene Pierce, HTL(ASCP)HT
Excalibur Pathology, Inc.
5830 N Blue Lake Dr.
Norman, OK 73069
405-759-3959
www.excaliburpathology.com

Re: de and restaining of histology slides
« Reply #4 on: October 24, 2010, 11:17:01 AM »