Hello, all.
 
I am doing double immunoflourescence in bone sections, but I find that there is so much background and there are so many spots, I have used the normal serum matched to the hosts of  secondary antibodies for blocking ( 5% donkey serum in PBS), spinined secondary antibodies over before incubating secondary antibodies and added washing times, but so much background still appears, I think that it isnot autofluorescence due to no background in negative sections, I am really sad because I can not find an effective method to reduce background. Any suggestions will be helpful! I am looking forward for your help!
 
Guofeng 



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