Dear Justin,

The best way to check is using a good positive control.

First titrate your secondary anitbody in combination with a primary antibody that has proven to work well (and with a clear staining pattern you are familiar with).

Than titrate your primary antibody on a proper positive control. If possible do a IHC staining on a section of the same tissue to check stainingpattern.

With kind regards,

Ronald

As Ronald suggested, this is one of those things you have to determine empirically.  Usually, when I am testing a new primary antibody, I'll try 3 different concentrations.  I usually look for the concentration where my specific signal no longer intensifies while maintaining a reasonably low level of background staining.  I always use the same concentration of secondary antibody (depending on supplier).  Without knowing more about your experiment, it's hard to give much more than that.  Good luck!

I think you might be using too much secondary antibody for your IF. I tend to use anything between 1:100-1:500 for my primary and 1:200-1:500 for secondary. By using too much secondary, you could be getting non-specific binding (resulting in a high background) - sounds like this is happening to you from your description.

As Ronald and Michael stated above, best to titrate out both antibodies to identify the best dilution to use. For both I would try 1:100, 1:200 and 1:400 and see in which combination gives you a good labelling - but as Ronald said, you need a good positive control for this.

Hello justin,

usually for experiment of IHC, ICC i usually use primary antibody ratio 1:100 and for secondary antibody i usually use 1:500-1:1000