36 days old neurons were stained with three different antibodies. The background was showed in the negative control also. However, for my colleague, who used the same antibodies in the same cells, there was only a slight background staining. Can anyone help explain why this discrepancy occurred? Is there something with the cell culture or staining?
Common to all is the secondary fluorochrome?
Looks to me it is aggregated and needs to be spun down to remove.
Did your colleague use a different secondary?
Hi Anjana Soorajkumar,
what antibodies are you using exactly? And what do you mean with negative control? Only the secondary but not primary?
If you coat with laminin, what animal species is it from (e.g. murine vs human)? If you have a secondary antibody against mouse this might detect mouse laminin.
There also might be something not working well with your blocking... but since the signal is so strong, like Carl Hobbs mentioned they look aggregated.
Negative control means using only the secondary antibody without the primary antibody. I used both mouse and rabbit antibodies. I need to check for laminin.
Thanks
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