2 replies to this topic

[dna_nerd]

I am using a mouse primary antibody on paraffin-embedded mouse sections (I know...) and experiencing high background.

I am altering my protocol based on some recommendations:

-Using TBS-Tween (0.05%) instead of PBS-Tween

-Adding 1% BSA to my blocking (I use 10% serum from the secondary species) and both antibody incubation steps, will increase time from 1/2 hour to 1 hour

It is also recommended to incubate sections in unconjugated AffiniPure Fab fragment anti-Mouse IgG (H+L) for 1 hour after the blocking step.  Either that or use F(ab) monomeric secondary antibodies.  Im a bit new to this and am not quite sure the biochemistry behind these steps so had a question:

I have a product in the lab that is Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block).  I believe this is just purified Fc receptors of mouse origin.  Could I use this product at all to help decrease background from Mouse on Mouse staining??

Thanks

[sgt4boston]

I am unfamiliar with that product but why not try it?  

It appears that the protocol requires you use antibodies devoid/missing the Fc component.  It could be that if the section contains tissue with Fc receptor then your specific ab will be bound providing you with a false signal (false positive).  

Another method could be to block the section with 'non-specific' normal mouse IgG for 30 min wash and then follow with your mouse specific ab?

Using the serum of one of the host species of your ab should cut down on non-specific binding (as you mentioned).

Good Luck!

[scolix]

we used this kit from Vector, for such immuno staining and it never gave us background when using mouse ab on mice tissues.