[Histonet] Double-staining with same secondary antibody?

[Min-Han Tan]

Dear colleagues,

I am planning a double stain of 2 antibodies - an endothelial marker and a
smooth muscle antigen on human tissue, and have some questions about
possibilities of using the same secondary antibody.

Primary antibody for endothelial marker = goat species; tissue requires
steam antigen retrieval.
Primary antibody for smooth muscle antigen = mouse species; tissue does not
require antigen retrieval. These bind to different areas of large blood
vessels - one to endothelia, the other to smooth muscle.

Currently, I only have immediate access to a biotinylated horse pan-specific
marker (anti-goat, anti-mouse), and I have separately optimized the 2
markers using this same secondary antibody.

Questions:
1. I wonder if it would be possible to pursue a double stain in a serial
(not concurrent fashion), with DAB-Nickel for the first staining to
"shelter", followed by NovaRed, using the same secondary antibody.

2. Is any elution step in between required, if I use the same secondary
antibody for the 2nd sequential antibody? Shouldn't the 1st primary antibody
be washed off, and in the worst case scenario, even if it isn't, will the
2nd red stain be obvious? :)

Am pending purchase of other biotinylated secondaries. Or should I just
purchase the DakoEnvision double staining kits?

Chris van der Loos' posts at
http://www.histosearch.com/histonet/Nov04A/HistonetRE.Elutionreagent.htmlwere
very instructive.

All advice would be appreciated!

Thanks.

Min-Han Tan