<< Previous Message | Next Message >>
From:Gayle Callis <> (by way of histonet)
To:histonet <>
Content-Type:text/plain; charset="us-ascii"

Saponin must be used in all buffers/diluents from blocking to substrate,
HRP or AP, then you need to CLOSE cell membrans by using a buffer rinse
WITHOUT the saponin BEFORE chromagen step and THEN develop the color.

For Cytokine staining on frozens, 0.1% saponin is recommended.
Found this to be fairly harsh on frozen sections,
required very brief and gentle rinse, with a pipette.

Tween 20, we use (to get rid of background) anywhere from 0.05%
to 0.2%, some may use higher concentrations.  Find 0.2% works quite well.
We haven't used Triton X100, but saw a protocol (routine IHC staining)
using Sigma's 10% Triton X100 diluted 5 ml/1000 ml buffer, fairly dilute.

Jules Elias book, Immunopathology, A practical approach to diagnosis
has an excellent discussion on detergents.

Hope this helps.

Gayle Callis

<< Previous Message | Next Message >>