I am injecting the hippocampus with an AAV8-GLT1 viral vector. I noticed when I am staining with immunofluorescence the secondary antibodies (Ms 598) is sticking to AAV virally injected tissue leading to false fluorescence drowning out the real signal. It almost seems like it is sticking to the areas where the virus transduced. How could I treat the tissue to prevent the secondary antibodies from sticking to the AAV?
First of all, you need to determine if it is a secondary or primary ab producing non-specific binding to the AAV8 related proteins to do so you need to incubate the sample with only secondary antibody. The other possibility is that your primary is non specifically binding to the AAV8. Also, you noted that you are using anti-mouse secondary meaning that your primary is of the mouse host. What is the host of material you are working with? Do you inject your AAV8 into the mice? If you have mouse material with anti-mouse antibody you may have the host conflict. Since ms secondary antibody recognizes the IgG there are IgG against your AAV8 vector produced by mouse while it is still alive, thus visualizing mouse IgG bound to the virus-infected cells visualizing it. The easiest solution would be to switch to the different species primary i.e. rabbit host primary to your target and rabbit secondary. Also, test your rabbit secondary for non-specific binding as described above by incubating samples after blocking with only secondary ab.
If it is the case
Never had this problem, but email me at s.waddington at ucl.ac.uk and we can send protocols
https://www.researchgate.net/figure/Immunofluorescence-of-mouse-brain-after-intraperitoneal-injection-with-AAV-hSLC2A1-At-3_fig3_312382933
Anton Lennikov Thank you for the information. I am having the same issue when I incubate only with secondary antibody therefore I do think it's the secondary recognizing something with the virus. I will try to switch the species to see if that makes a difference.
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