The rabbit anti-Fluorogold from Chemicon (AB153) used to work okay with a fluorescent secondary, but they don't make that anymore. I've tried their replacement (AB153-i), and also Fluorochrome's antibody, but neither primaries are working.
Thanks in advance!
I've gotten the replacement (AB153-i) to work decently, though a higher concentration was required - 1:10K for the old version, 1:2k for this new version. Alternatively, Protos Biotech (www.protosantibody.com) has a decent antibody for fluorogold. It requires an even higher concentration (around 1:500 in my hands). The protos antibody is made in guinea pig, which can be helpful for multiple labeling but annoying due to needing another secondary antibody.
Originally, I had trouble using the old AB153 on 4% formaldehyde-fixed tissue, stained free-floating style - there was virtually no staining on top of the native UV-excited fluorogold fluorescence. However, the IHC staining signal became very robust, moreso than fluorogold's native fluorescence, after pepsin-based antigen retrieval. Such a procedure may be needed. I recommend NeuroMAb's protocol, linked below.
http://neuromab.ucdavis.edu/files/Ag.pdf
I've gotten the replacement (AB153-i) to work decently, though a higher concentration was required - 1:10K for the old version, 1:2k for this new version. Alternatively, Protos Biotech (www.protosantibody.com) has a decent antibody for fluorogold. It requires an even higher concentration (around 1:500 in my hands). The protos antibody is made in guinea pig, which can be helpful for multiple labeling but annoying due to needing another secondary antibody.
Originally, I had trouble using the old AB153 on 4% formaldehyde-fixed tissue, stained free-floating style - there was virtually no staining on top of the native UV-excited fluorogold fluorescence. However, the IHC staining signal became very robust, moreso than fluorogold's native fluorescence, after pepsin-based antigen retrieval. Such a procedure may be needed. I recommend NeuroMAb's protocol, linked below.
http://neuromab.ucdavis.edu/files/Ag.pdf
Hi Kevin,
I've tried the AB153-i at 1:2k, but not with pepsin antigen retrieval. I'll give it a crack this week. Thank for the tip!
Cheers
Jamie
Hello Jamie,
Did you try the pepsin digestion protocol? How did it work? I also use AB153-i without much success...
Thanks!
Ronan
@Jamie, did you try the AB153-i staining without pepsin antigen retrieval and does it work?
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