I'm having a heck of time! I'm trying to perform triple immunofluorescence for CAMKII (alpha; mouse; milipore 05-532; 1:5K; visualized with AlexaFluor 647), Fluorescein (Invitrogen, A11095, goat, 1:500; visualized with AlexaFluor 488) and Fluoruby (Molecular Probes, A6397,rabbit, 1:1K; visualized with AlexaFluor 568) in the macaque brain. Each works beautifully by itself. FR and FS work beautifully together. However, when I put them all together, I am unable to resolve the CAMKII staining. I have processed all primaries together and sequentially. Primaries have been incubated from 1 night up to 4 nights (different trials), the Fluors have been incubated overnight (at room temperature or in the cold room shaking). All primaries are made in different animals (goat, mouse, rabbit) and blocked in a donkey serum block. The fluors are Donkey anti-Fluor. Any and all help would be great appreciated! Thanks!
Hi Emily,
Sorry to hear you're going through some teething issues. This is probably not much help but are the fluorescein and fluoruby just tracers? If so can you visualise it without amplification with the AF488 + AF568 is it worth just doing the CaMKII immuno and adjusting the exposures accordingly?
If not as you mentioned, as the CamKII antibody is working well perhaps if you react that first and confirm with visualising under the microscope then you can go back and amplify the tracers afterwards.
Good luck with your experiments.
- Badges
- Science topic