Which is the best way to performe multiple labeling (dual staining) IHC with two mouse monoclonal Abs, to detect two molecules in the same cell compartment? Is this possible or is better to try IF?
When you say two molecules in the same cell compartment, then they are very very close to each other, much shorter than complex that precipitates during DAB reaction. Immunoflourescence is recommended here as it will make life much easier, but if that is not an option then one may think to reduce the primary antibody concentrations to the extent that you see both HRP and AP signals distinct from each other within the mentioned cellular compartment. Optimization of primary antibodies without compromising quality can do the trick. Good Luck :)
Hi Luis,
The way to go, mainly because you are using two monoclonals, is IF; this will give you the less headaches. But if not possible, try the recomendation of Dr. Lakshman Puli with the kit sugested by Dr. Drndarevic. One more time, the way to go is IF. Good luck.
Dear Luis
All the repies are valuable but you have to consider that both of your Abs are mouse monoclonals and you can not use them to gether nevertheless you use Abs in this order
1-First primery Ab exp Anti A
2-Conjucated secondry Ab exp goat anti mouse FITC
3-Second primery Ab exp B
4-Conjucated secondry Ab exp goat anti mouse Texas Red
because if donot do this in this manner your secondry Abs will be attache to the primry Abs randomly
Regards
You can either use directly labeled primary antibodies (if available) or stain subsequently but include a blocking step in between (1st prim. ab, 1st sec. ab, blocking, 2nd prim. ab, 2nd sec. ab). The blocking has to be done as follows: 1h 10% mouse normal serum followed by 1h Fab anti-mouse (unlabeled). It is necessary to prevent binding of the 2nd sec. ab to the first prim. ab.
hope, this helps. anja
i recomande you to try the new product from Dako the Envision DuoFLEX Doublestain
Dear luis,
I'm having the same difficulties as you were having. I have two mouse monoclonal Abs which I woul like to dual stain with IHC-P. Have you figured out a method? Or is it only possible to do this with IF? I was looking at the Animal research kit of DAKO. Maybe this is an option as well?
Best regards
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