Dear So-Jin

From what you tell, specially regarding with signal in green and red channels in untagged samples, I'd bet you have an issue with lipofuscin autofluorescence. Lipofuscin has been reported to have very high levels in liver. Check out the papers I'm attaching, the magnesium acetate buffer from the Schnell paper worked out very well in my adult mouse brain slices. Be aware that you "might" have to reset your antibody concentrations after these treatments specially with Sudan Black.

Good luck!

Try to use fluo label with higer emission, if you can do it with your equipment. Check the detector range too.

Dear Sebastián

Thanks for your helpful comments.

I will try to hire the treatment of magnesium acetate in my liver tissue.

Have a nice day :)

Dear Attila

Thanks for your helpful comments.

I will check the detector range. Frankly I never think about the detector range before. 

Thank you :)

I recommend that you verify your findings with an alternative method (e. g., Western-Blot analysis using specific antibodies for detecting autophagy marker proteins).

Best regards, Christian

I had a similar problem. My unstained tissue (plant-tissue) showed a red fluorescence nearly similar to my fluorescence-antibody. By using the Lambda-scaningmode of the laser-scaning-microscope one could verify the emission spectra of every single signal to determine if it is autofluorescence or not. In my case, I found signals with an emission spectra matching to my antibody.

Hello

Agree with Attila Lehotzky ,  use fluo label with higer emission

Find the papers here , may help you

Good Luck 

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2989884/

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2669416/

http://www.nature.com/ncomms/journal/v1/n9/full/ncomms1144.html?message-global=remove

http://ajp.amjpathol.org/article/S0002-9440(15)00332-6/pdf

Dear So-Jin, in our lab we do immunofluorescence daily and we have lot of experience with antibodies and non specific signals. It really depends on tissue, antibody concentration and quality of course. I am sending you one file which might help you: it s about microwave heating and different antigen retrieval methods that we use! ;) As Christian Q. Scheckhuber said, Western blot is always good choice ;) Good luck and all the best from Croatia

I agree with Subrot mentioned the controls, especially isotype for primary Ab. More or less the primary Ab will produce nonspecific fluroscence. For some tissue which express high FcR, it is very common to get false positive signals without isotype control. Just as isotype control in FACS, gain and offset setting on Confocal can be made on isotype control group, then the signal we have in experimental group should be real.