I injected my rat via intravenous tail vein injection with an AAV2-CBA-GFP virus... waited 14 days for viral infection and then perfused my rat with PBS to wash out the blood and 4% PFA to fix all tissues. Post fixed the liver overnight in 4% PFA made in 0.2M PO4, sucrose cryoprotection, embedded in OCT and sectioned 10 µm sections.
When i was performing GFP staining with anti-GFP primary antibody and Alexa-488 conjugated secondary antibody I could not see any GFP signal in my green channel. All cells were fluorescing green, even in my negative control sample where I did not inject the virus.
I used 0.3M glycine to quench during my IF protocol.
Can anyone help me in reducing background fluorescence in the liver?
In my guess, you may need more washings after secondary antibody and also reduce the concentration of secondary antibody (i.e., make more diluted). At least control should not give staining.
TrueVIEW Autofluorescence Quenching Kit works well for liver tissue. Good luck!
Organ autofluorescence is a major issue. Most modern methods to avoid this problem involve making organs transparent. I hope the inserted articles are of use to you. Best of luck!
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