I am doing immunohistochemical staining with DAB development on rat brain tissue. The first immunostainings with rabbit anti-alpha-synuclein and rabbit anti-GFP antibodies went well and look as expected. However, when I stained with mouse anti-TH and goat anti-VMAT2 I get a very high background even though I am using the same protocol. It looks like there is a lot of unspecific binding.
Blocking is done with 5% appropriate serum for 1 hour at room temp. Incubation with primary antibody in 2.5% serum overnight at room temp, and secondary antibody in 1% serum for 2 hours at room temp. For the alpha-synuclein and GFP antibodies goat serum was used (secondary was goat anti-rabbit), and for the TH and VMAT2, horse serum was used (secondary was horse anti-mouse and horse anti-goat, respectively).
Does anyone have any suggestion as to what may be the cause of this problem? Maybe someone experienced a similar problem?
-You can increase your blocking time to overnight in 4C
-You can increase your washing steps
-You can reduce DAB exposure time
-If your primary antibody is unconjugated you can use florecent secondary antibody if the confocal microscope is available.
-If nothing works try to change primary antibody to a different company maybe this specific strain recognise some non-speicific epitope in brain tissue.
Thanks for the advice. No, I did not do antigen retrieval for the TH or VMAT2 staining. The strange thing is that these are all routine immunos in our lab and normally work very well with our protocol. Other people in the lab are doing the same stainings on rat brain tissue as well and do not seem to have a problem with high background.
I will try to do heat-mediated antigen retrieval and also with BSA for blocking instead of horse serum.
CAS-block is also good for blocking. It solved our background problem some years ago.
Thank you :-)
The animals were transcardially perfused with 200 ml saline followed by 150 ml 4% PFA. The brains were then post-fixed in PFA for 3 hours at 4°C, cryoprotected in 25% sucrose solution and sliced.
Hi Josefine,
You could try incubating the sections in primary antibody at 4 degrees for overnight. What concentration of secondary antibody are you using? if you are using very high concentrations of either primary or secondary antibody, it would increase your background staining. You could also increase the blocking duration to either 2 hours at room temperature or overnight at 4 degrees. Hope it helps! Good luck.
I tried to do antigen retrieval and it completely removed the background from the TH staining!!
Thank you all for the advice.
It could be high concentration of abs. Try lower concentrations/
Hi Josefine,
Great to hear that antigen retrieval solved your problem, but just in case other people have similar issues: overnight incubation is probably more reliable on 4°C instead of room temperature, and getting rid of serum altogether from the primary antibody mix has also helped us minimizing background.
I am having a similar issue while using frozen tissue. I have been trying to optimize a rapid (1.5 hr) stain on human brain tissue and am experiencing a lot of non-specific binding. I went through the protocol and did not use any primary or secondary antibody to try to find the issue, but even without antibody, I still have non-specific staining. I am also using DAB. Has anyone experienced the same issue?
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