Currently, I am working on CD86, CD11b, CD45, and F4/80 IF staining in DRG tissue, but I believe CD86 and CD45 are binding non-specifically. Instead of staining specific areas, it appears as scattered dots across the whole tissue. I tried lowering the Triton X-100 concentration from 0.3% to 0.1%, but the issue persists. For fixation, I used PFA for 12-16 hours. The primary antibodies for CD86 and CD45 were both used at a 200:1 dilution. Previously, I tried different antibody combinations, but the same issue occurred. I then thought that the antigens might be masked during the fixation process, so I tried sodium citrate for antigen retrieval, but this damaged the CD11b staining. I’m unsure which part of the process to modify to improve the specificity of the staining. Any troubleshooting advice would be greatly appreciated
Hello, Hyeyeon!
DRG tissue staining tends to exhibit nonspecific staining (similar to brain tissue), as the ganglia can adsorb charged molecules. Therefore, it is recommended to use mild antigen retrieval during the repair process. Additionally, CD86 is not expressed by all immune cells. If you are performing multiplex staining, it is advisable to stain for CD86 first and include a positive control to rule out potential issues with the antibody itself. CD11b and F4/80 can be stained afterward. Since CD45 is expressed by all immune cells, it can be stained last. For conventional tissue samples, both EDTA and citrate can be used for antigen retrieval. However, if sodium citrate buffer interferes with subsequent staining, you may opt for EDTA instead. It is recommended to weaken the retrieval conditions and try microwave heating at medium or low power.
Hope this helps you, you are welcome to engage in more detailed discussions.
The potential problem here is two-fold:
1. Historically and commonly, antibodies against CD antigens have been used for LIVE (not fixed) cells from blood or other biological fluid. So many the famous CDs antibody only works for live cells. You need to check in the datasheet whether it works with fixed sample.
2. CD antigen also membrane bound, so Triton X-100 can cause the antigen to solubilize away from the membrane. Try omiting the Triton-X or use saponin based stainig buffer if you want to co-stain with internal antigen.
Fixation 12-16 hour it seems to long if done in RT and small tissue size.
- Badges
- Science method
- Similar topics
- Biological Science
- Ecology