I stained various markers (CD3, CD8, CLEC4F, etc) in liver tissue containing metastatic tumor nodules for immunofluorescence imaging, but when I tried to take a picture showing tumor region and liver region together in one frame, there were too many non-specific background fluorescence in liver region. I tried adjusting the fluorescence to get rid of the background staining, but adjusting it based on liver tissue made positive staining in tumor region fade away. (I attached an image for your reference) There was no such problem when I stained the tissue with TUNEL and DAPI, which both stain DNA.
It seemed like autofluoresence and non-specific binding could be the problem, so I am trying to redo the experiment in perfused liver tissue (containing metastatic tumor nodules) and also change blocking solution (From 5% BSA + 0.3% Triton X-100 in PBS to 1% BSA + 5% Normal serum + Glycine + 0.3% Triton X-100 in PBS, RT for 2 hours).
I was wondering if anyone else has also experienced the same problem when staining liver tissue for IF imaging. If so, could you please share how you handled the problem?
Thank you!
Hi,
liver is just one of those tissues that is known to high a very high autofluorescence. I would suggest staining your markers with a PE or even preferably APC fluorochrome as then you can simply filter out the signal in FITC as autofluorescence.
I also worked with LC3-II once, in highly AF tissue and I quenched this by using an incubation with Sudan black. Maybe read this attachment.
Best of luck!
Cristina
Article Optimization of Single and Dual Color Immunofluorescence Pro...
- Badges
- Science topic
- Similar topics
- Chemistry
- Pharmacology
- Medical Pharmacology
- Clinical Pharmacology