Re: TEM immunolocalization problems

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From:Tom Kuwahara <> (by way of histonet)
To:histonet <>
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Dear Dwight:  Long ago when I was doing IHC in JB4 embedded specimens, I
found that over a relatively short time( less than a month) I lost my
antigenicity in the block.  Keeping the blocks in the freezer helped a
little bit but mostly I had to cut a lot of sections initially and these
seemed to hold their reactivity better than the whole block.  I'm sure
someone out there in Histoland can give us a better explanation of what
happens or whether or not this also happens in LR White blocks.
Probably something to do with the resin continuing to polymerize over
time.  I don't know if you can recover your AB or not.  Anybody out
there know?  Regards, Tom Kuwahara

Dwight Beebe wrote:
> Good morning everyone,
>         I have encountered considerable difficulty, i.e., zero success, in
> repeating an immunolabelling experiment.  I'm working with LR White
> embedded cotyledon tissue, fixed 12/95, probing with a polyclonal Ab
> (rabbit), also made in '95.  My secondary is commercial protein A gold.  I
> had very specific labelling when the initial experiment was conducted in
> '95, but now have no specific label.  I have repeated the previous
> successful protocol, but with no luck.  My antibodies have been at -20C,
> having only been thawed once to aliquot them into smaller volumes.
>         Is it possible that there have been changes in the fixed tissue
> that cause loss of epitopes?  I find this hard to believe, but I'd like to
> hear from others.  Could the antibodies have lost their specificity?  I
> have repeated dilution series experiments, but again, all labellings were
> no more specific than buffer controls.  I should say that the antibodies
> work fine in Westerns, giving us the same banding pattern we've always seen
> with these Ab.
>         Any and all comments, criticisms, speculative remarks, etc. would
> be most welcome.  Much hinges on the success of the labelling.
>         Many thanks,
>                 Dwight
>Dwight Beebe
> Prof. Agrege (Associate Prof.)
> Institut de recherche en biologie vegetale
> Universite de Montreal
> 4101 est, rue Sherbrooke
> Montreal, (Quebec) H1X 2B2 Canada
> Tel: 514/872-4563 or -4746 (lab)
> FAX: 514/872-9406

Thomas J. Kuwahara
Senior Immunohistochemist
Advanced Pathology Systems
3801 Sacramento St. suite 621
San Francisco, CA 94118
415 750 6800 x23067 tel
415 750 2332 fax

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