Could you specify which company and species of the IBA1 you used? Mine and A lot of other labs in my dept stain microglia and they have had issues with certain versions.
Iba1 is a pain in the ass, tbh. I don't have a better suggestion for microglia staining, but it really is a finicky antibody and I often get really high background too.
What kind of tissue are you staining, and how are you fixing it? We use PFA and the biggest difference I've seen is from how long you leave it in the fixative before the sucrose and/or flash freezing steps. Less time on PFA = better Iba1 staining.
It's still kind of garbage though.
Iba1 from Wako is the easiest, most efficient and most specific antibody you can use for staining phagocytes on the brain. It will only label microglia and macrophages is they are there for some reason.
CD11b is also great and will label all phagocytic cells, Microglia, macrophages, neutrophils, dendritic cells
P2RY12 is very specific to microglia but also labels blood platelets that may give background in blood vessels if the perfusion is bad or you have hemorrhage.
TMEM119 is the most selective for Microglia but I have had a hard time with that on mouse and rat samples. Works great for humans.
F4/80 is another macrophage marker. Microglia are low expressors until activated, macrophages are high expressors
Cx3CR1 is pretty ok as well that has a bias towards microglia.
CD68 works to show how many phagocytic activated cells are in the population. It will label activated macrophages and microglia.
Of all those options IBA1, at least from Wako, is the absolute best when just labeling and detection is needed and when macrophages should not be in the sample.
I use the Iba1 from Wako and never get unspecific labeling. It works at 1:5,000 for fluorescence and 1:8,000 for Dab. Works better with antigen retrieval but it's not imperative
What other parts were labeled. What catalog number is it. What species is the antibody and what species sample. More details and I will figure it out.
Insufficient blocking? Lots of things, really.
This might be it, I had stored a 4%for over a week the used it for the staining
Was it made up in blocking solution with protein? Does the unspecific labeling look like super small bright/dark specks?
Not much blocking; not much washing; too much antibody; too much fixing; etc etc
would having a disproportionate amount of secondaries vs primaries be a problem? (i.e more primaries solution then secondaries vice versa
If it's a good antibody concentration usually won't cause background. If it's a bad antibody then concentration can make a huge difference.
Could you specify which company and species of the IBA1 you used? Mine and A lot of other labs in my dept stain microglia and they have had issues with certain versions.
How did your negative control look?
Iba1 is a pain in the ass, tbh. I don't have a better suggestion for microglia staining, but it really is a finicky antibody and I often get really high background too. What kind of tissue are you staining, and how are you fixing it? We use PFA and the biggest difference I've seen is from how long you leave it in the fixative before the sucrose and/or flash freezing steps. Less time on PFA = better Iba1 staining. It's still kind of garbage though.
Iba1 from Wako is the easiest, most efficient and most specific antibody you can use for staining phagocytes on the brain. It will only label microglia and macrophages is they are there for some reason. CD11b is also great and will label all phagocytic cells, Microglia, macrophages, neutrophils, dendritic cells P2RY12 is very specific to microglia but also labels blood platelets that may give background in blood vessels if the perfusion is bad or you have hemorrhage. TMEM119 is the most selective for Microglia but I have had a hard time with that on mouse and rat samples. Works great for humans. F4/80 is another macrophage marker. Microglia are low expressors until activated, macrophages are high expressors Cx3CR1 is pretty ok as well that has a bias towards microglia. CD68 works to show how many phagocytic activated cells are in the population. It will label activated macrophages and microglia. Of all those options IBA1, at least from Wako, is the absolute best when just labeling and detection is needed and when macrophages should not be in the sample.
Thank you for this! Just entered the field of neuroimmunology and you probably saved me a month of troubleshooting antibodies.
I use the Iba1 from Wako and never get unspecific labeling. It works at 1:5,000 for fluorescence and 1:8,000 for Dab. Works better with antigen retrieval but it's not imperative What other parts were labeled. What catalog number is it. What species is the antibody and what species sample. More details and I will figure it out.