problems with immunolabeling
Mark H. Whitnall
whitnall at vax.afrri.usuhs.mil
Wed Oct 25 10:47:06 EST 1995
An avidin-biotinylated peroxidase tag is much more sensitive than
PAG and will allow detection of epitopes where PAG fails.
The DAB reaction product stays localized to the site where the
antibody binds. See Exp. Cell Res. 216:411-421 (1995) for a
pre-embedding protocol and J. Comp. Neurol. 275:13-28 (1988)
for a post-embedding protocol.
> I am currently having difficulties with poor labeling on S. aureus
> and S. epi cells. The antibodies have been tested by ELISA and the titers
> are very high 1:50,000. The cells are embedded in LR White and ultrathin
> sectioned. After labeling the cells are viewed by electron microscopy. I
> have tried several protocols, including using skim milk and BSA to block with
> at different concentrations. Additionally the PAG binds nonspecifically to S.
> aureus and thus IgG goat anti-mouse or anti-rabbit must be used as the
> secondary antibody. I have tried these for a variety of concentrations and
> times. The primary antibody has been used at a concentration of 1:20, in which
> there is no labeling, to 1:5, in which the labeling is small. Thanks for your
> help. If you need additional info please e-mail.
Mark H. Whitnall, Ph.D., Armed Forces Radiobiology Research
Institute, Bethesda, MD 20889-5603 tel: (301) 295-0277
fax: (301) 295-0313, whitnall at vax.afrri.usuhs.mil
http://22.214.171.124 gopher, ftp: 126.96.36.199/
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