We've been working to incorporate BV421 and BV480 into our fluorescence imaging workflow. While both secondary antibodies (Jackson Immunoresearch) work well at labeling targeted antigens, we've encountered an issue where, after a day or two, the BV421 fluorescence has diffused into adjacent tissue (from pancreatic islets into acinar). BV480 remains where it should.
So far, we've mounted in Vectashield Vibrance and Prolong Diamond. We'll try basic aquamount and Prolong Glass next.
Anyone else had this issue? I'm thinking we may try doing a brief fix in PFA after secondary labeling to see if that helps bind the secondary but if this is due to dissociation of the BV polymer from the antibody I doubt fixation would help much.
Update: As mentioned in the original post, we tried a post-fixation step after secondary staining (10 min RT with 10% NBF) AND mounted the slide with Lerner Laboratories Aqua-Mount. The staining was nice immediately after mounting. Kept the slide @ 4C for 4 days and checked the location of BV421 stain. All was good. Beta-cell specific staining was still intact with no dissociation of the fluorophore into the surrounding tissue. We will now test Aqua-Mount alone (no NBF fix) as well as NBF fixation prior to Prolong Glass and Vectashield Vibrance mounting to see if this is due to the mounting media alone. I'll update when I have the results.
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