Keith,
Do you need fluorochromes on these sections? If not then I would
recommend the following:
1.Block Non specific sites with normal sera.
2.Incubate primary #1.
3.Wash
4.Incubate secondary HRPO conjugate
5.Wash
6.Apply a permanent chromogen such as Di amino Benzidine, gives
a nice Brown colourat the reaction site.
7. Remove the first primary using either enzyme (We use Protease XIV
0.05 mg/ml) or a dilute acid solution (I think a 0.1 N HCl for 10 min
should do the trick) Remember to use poly lysine or silaned treated
slides or your sections will come off.
8. Reblock with 5% normal sera
9.Go through with the second primary only this time use a chromogen
like AEC which although it is soluble in organic solvents gives a
nice red end product. Counterstain with Hematoxylin and coverslip
with a water based mounting media.
A couple of caveats to the above;
It may be that one or both of your antigens do not survive the elution
process. If it does not work one way it may be that it might work if th the second antigen is stained first.
If neither one survives the elution process, and you are working with
frozen sections you could try a brief (10 sec) "fixation" step prior
to the elution using 0.05% paraformaldehyde which may stabilize the
antigen. This tends to be tricky because the immunoglobulins are also
fixed to the section, it may be best to try the fixation prior to any
staining, so much depends on the nature of your antigens you are trying
to detect. We have done this with a number of infectious agents and
some cell surface markers on formalin fixed tissues, but it is not
my favorite pastime. Good Luck.
Regards
BJC - Brian
The spark of life is to accepted as a sacred trust to be transmitted
undimmed to future generations.
