anyone knows good HA antibody for zebrafish whole mount stainging - (Oct/27/2008 )
I am doing HA -tag antibody staining in zebrafish embryos and suffered great difficulity. I already used HA antibody from Roche, 3F10 anti rat monoclone . anti-mouse from Babco MMS-101p, anti-Rabbit from Santa cruz. None of them worked .
Does anyone here know good HA antibody for zebrafish whole mount antibody staining?
I'd really like to help since I work with HA antibodies all the time but I've never done a whole mount before. I use the 16B12 monoclonal for IF on cultured cells and get great results. Invitrogen (Molecular probes) has a primary conjugated to alexa fluorophores. This is optimal for any type of staining since you won't have to use a secondary and might be a good one for you to try.
Thanks ekay,
I also used 16B12, it did not work. Thank you for your suggestion, it is worth trying conjugated antibody from Mol. Probe.
I also used 16B12, it did not work. Thank you for your suggestion, it is worth trying conjugated antibody from Mol. Probe.
The only other thing I can suggest is trying the chicken antibody from aves lab. I only used it in a western blot (after an IP with rabbit anti-HA) but it was by far and large the cleanest HA antibody I've seen. They also have FITC conjugated as well. The data sheet says it's good for western and IHC so it may work for your zebrafish. Just curious, when you say the other antibodies aren't working, what specifically do you see? No staining? Everything non-specifically stained? I'm just thinking that your problem may not necessarily be with the antibody.
I also used 16B12, it did not work. Thank you for your suggestion, it is worth trying conjugated antibody from Mol. Probe.
The only other thing I can suggest is trying the chicken antibody from aves lab. I only used it in a western blot (after an IP with rabbit anti-HA) but it was by far and large the cleanest HA antibody I've seen. They also have FITC conjugated as well. The data sheet says it's good for western and IHC so it may work for your zebrafish. Just curious, when you say the other antibodies aren't working, what specifically do you see? No staining? Everything non-specifically stained? I'm just thinking that your problem may not necessarily be with the antibody.
Hey rKay,
Actually I injected HA-tag mRNA into zebrafish embryos ,fixed the embryos and did the immunostaing.
I took the un-injected embryos as negative control. I observed some weak signal actually, like weak staing in cytosol in both injected and unjected embryos, there is no signal difference betweem them therefor I said it is unspecific staining.
I also another antibody from Abcam today, it is expensive. It works well with culture cell in ICC. Hope it works in zebrafish embryos.
Thank you for your suggestion