|From:||"John C. Dennis" <firstname.lastname@example.org>|
I've compared triton x 100, tween 20, and saponin on tissue sections and
seen no difference whatsoever with any of the three when compared to no
detergent. At least not with the antibodies I've tested. I believe a mAb
to GAP43 was the only one that called for saponin (in a protocol I
inherited). Certainly in paraffin embedded tissue, I can't see any point
in using detergent unless the target is some truly funky
epitope. Detergents make my frozen sections come off the slides
(poly-L-lysine coated) far more readily so I've quit altogether. Lately,
though, I've had either some low quality antiserum OR I should go back to
using some detergent. We'll see.
You mentioned running a comparison among several antibodies to IFNgamma
and finding that some published facts are nonsense. Could you elaborate
just a bit without embarrassing anyone?
John Carroll Dennis
Anatomy, Physiology, and Pharmacology
109 Greene Hall
Auburn University, AL 36849
On Wed, 20 Dec 2000, C.M. vander Loos wrote:
> >I have heard mention about using saponin for cytokine immunohistochemistry
> >(ie. Interleukins, etc) on formaldehyde fixed paraffin embedded material.
> >Has anyone more information. Is it used in the wash buffer or retrieval
> >Jim Manavis
> Saponin is very useful for immunostaining of cytokines in intact cells.
> Saponin creates holes in the outer membrane allowing your antibodies go in
> and out. The action of saponin is however reversible. After after washing
> with buffer without it, the cells will close again. Therefore, you need to
> add saponin to ALL reagents from endogenous peroxidase blocking until the
> chromogen solution.
> Although there are reports that claim its necessity the use of saponin for
> tissue sections is however doubtful. Personally I believe that after
> sectioning of a tissue block, all cells in it are cut through, having their
> interior completely exposed. From this point of view saponin is not needed.
> However, you may just add it as a "soap" for reducing non-specific binding
> of your antibodies, like with Tween-20 or Triton-X-100.
> Recently, we have completed an investigation to the reliability of IFNgamma
> immunohistochemistry and cytochemistry using 13 different commercially
> available antibodies. It came out that at least with anti-IFNgamma on
> tissue sections a lot of nonsens has been published!! It is our impression
> that this situation is certainly not unique for IFNg...... So please be
> careful with cytokine immunohistochemistry reports!!!!!!!!
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