First thank you all for your helpful responses! Now another problem
In <s.tout-170394194416 at 220.127.116.11> s.tout at cclru.unsw.edu.au writes:
> In article <2m463l$pe0 at wega.rz.uni-ulm.de>, CCC_GAME at rzmain.rz.uni-ulm.de> (Gamel Anton) wrote:
> ...stuff deleted... double labelling with two monoklonals from
> > mouse differing only in isotype: IgG1 vs. IgG2b. There are lots
> > of conjugated polyklonals that discriminate IgG1 and IgG2b ...
> > The experiment should show the two antigens in one cell.
> > There are some monoklonal 2nd ABs (only one red) available.
> > What system should I use monoclonal or polyclonal...
> You should be able to get specific monoclonal- 2nd antibodies for your
> different isotypes, just make sure on the technical description that
> they're affinity purified and don't cross react. Have you also considered
> 2nd antibodies to the light chains of your primaries?
Oh, no! I discovered that this only "orange-red(!)"-conjugated
monoclonal antiMouseIgG is one with R-Phycoerythrin that is mainly used in
Flow Cytometry not in Immunohistochemistry, emissing a peak at 575nm
- the specific red filter blocks if lower than 580-590nm!
I suppose I can see *both*, FITC *and* R-PE, with a 520nm filter but the
pictures should be separated e.g. for b&w photos. Additionally
recommended dilution is 1:20 - 1:40 :-(
Now we try to go for a polyklonal intermediate antibody from rabbit
that is adsorbed to other IgGs and shows "minimal cross reactions"
to goat antibodies (DAKO 0.1"%" cross reactions in a scale of 0.1
(not detectable) to 10% (severe cross reactions)).
Steps are now:
1. Mouse monoclonal IgG1
2. goat anti (H+L) mouse, conjugated
this is a normal indirect labelling... second labelling as follows:
3. Mouse monoclonal IgG2b
4. rabbit anti IgG2b mouse, unlabeled
5. goat anti (H+L) rabbit, conjugated
The rule is: if possible take second antibodies from one species
:-( this is not possible here.
Are there any objections to this system??
Anton J. Gamel
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