Hi Lauren E Fritsch ! I routinely do free-floating IHC on neonatal rat brain sections. I usually do co-labeling for confocal microscopy, and end up using several different antibodies from various hosts. I have found that a blocking buffer of 10% horse serum/10% goat serum/PBS/0.05-0.1% tween almost always gives me a very low background. Since most secondary species are goat/horse/donkey this blocking formula works very well. Even if I am not co-labeling I often still use this block, and it works like a charm! I will say that this is a pretty high overall concentration of sera, but it works really well for me 98% of the time.

However, I have an antibody I'm optimizing right now and it performs better with 5% BSA/PBS/0.1% tween for whatever reason (though secondary is goat anti-rabbit), so sometimes it will just take optimization and trial and error. I have also found that a high serum concentration in block does not always perform quite as well when using DAB as a chromagen, but is great for my fluorescence. So overall, I'd say stick to the old rule of thumb and first go with serum from the host of the secondary, in a concentration of 5-20%. You can also try my cocktail especially if you're doing florescence and/or co-labeling. And sometimes things are weird and it may take BSA or even nothing at all. And finally, if you're routinely experiencing a lot of background I HIGHLY recommend pre-absorbed secondaries - they are worth the money! Good luck!

Hi Lauren E Fritsch ! I routinely do free-floating IHC on neonatal rat brain sections. I usually do co-labeling for confocal microscopy, and end up using several different antibodies from various hosts. I have found that a blocking buffer of 10% horse serum/10% goat serum/PBS/0.05-0.1% tween almost always gives me a very low background. Since most secondary species are goat/horse/donkey this blocking formula works very well. Even if I am not co-labeling I often still use this block, and it works like a charm! I will say that this is a pretty high overall concentration of sera, but it works really well for me 98% of the time.

However, I have an antibody I'm optimizing right now and it performs better with 5% BSA/PBS/0.1% tween for whatever reason (though secondary is goat anti-rabbit), so sometimes it will just take optimization and trial and error. I have also found that a high serum concentration in block does not always perform quite as well when using DAB as a chromagen, but is great for my fluorescence. So overall, I'd say stick to the old rule of thumb and first go with serum from the host of the secondary, in a concentration of 5-20%. You can also try my cocktail especially if you're doing florescence and/or co-labeling. And sometimes things are weird and it may take BSA or even nothing at all. And finally, if you're routinely experiencing a lot of background I HIGHLY recommend pre-absorbed secondaries - they are worth the money! Good luck!

Hi Laure, I absolutly agree with Katie's recommandation. I have made more or less the same experience . One thing I have to add, is the problem when you use mouse antibodies on mouse tissue. If it is possible take the primary out of different species. If not take 20% normal serum in the block as well as in the delution medium of your primary antibody. If you are working with streptavidin as a detection system you have to block the endogenious biotine (Vector Labs offer a suitable blocking kit). And last but not least if you are trying differne blocking protocols use negative controls (no prim. antibodies). Negativ controls show you all unspecific background staining of secondary antibodies, detections systems , used normal sera or BSA as well as detergents like tween20 or Triton-X100.

Thank you! I tried 20% goat serum and got great signal, but it looks like there's a bit of background that I think I could reduce. I'm planning on testing it on another genotype this week and will also try a negative control to confirm the background before trying another blocking buffer. Appreciate the help!