We have conducted an experiment where mice were injected with Evans Blue (i.v.) and then perfused 1 h later. Brains were extracted, hemisected, and one half was post-fixed (4% PFA, 48 h) for sectioning.
As Evans Blue autofluoresces red, we decided to perform DAB staining to look at various markers (GFAP, CP13, etc..). However, we find non-specific vascular staining with the DAB, even in our no primary controls.
I am assuming that this is the EVB reacting somehow with our protocol - has anyone else seen this? Does the EBD bind the secondary antibodies?
The protocol we use is as follows (on 40um free-floating sections):
10 mins TBS wash, 20 mins hydrogen peroxide (0.6%) quench with Tx-100 (0.1%), TBS washes, 1 h block in 10% FBS in TBS-Tx 0.1%, Primary incubation in 2% FBS in TBS-Tx 0.02% (o/n), TBS washes, Secondary incubation in 2% FBS in TBS-Tx 0.02% (2 h), TBS washes, ABC incubation (45 mins), PBS washes, DAB reaction, mounting & dehydration.
Any comments would be greatly appreciated!
Thanks for your answer Baptiste, those are good suggestions.
This is a well established protocol in the lab, which is why this staining was strange. We usually do not see any non-specific binding with 0.6% H2O2 in any of our perfused brains. We did a control experiment on brains that had no EVB and they did not show the same staining but we will definitely try higher concentrations in these mice to see if this reduces the background.
If this is an established protocol and the problem is new, I suggest that perhaps one of your reagents has become contaminated, gone bad, or was improperly prepared recently. Take a close look at any and all things that are different between the unexpected result and your most recent normal result.
A lot of acededmic laboratories do nto keep very good records, but if you are lucky and your lab adheres to GLP standards, you should have lot numbers of reagents recordedde as well as the preparation date and initials indicating who prepared reagents that are prepared in house, so you can easily figure out what has changed since the last successful experiment. I highly recomment keeping track of manufacturer and lot number for all reagents as well as prepared dat for in house reagents. It really simplifies when something unexpected happens.
Good luck!
Hi Amy,
Have you tried ABC/DAB staining of the Evans Blue sections without *any* antibody (primary or secondary)? and DAB staining without the ABC or the antibodies? Are you using any Ni2+ enhancement? Just trying to isolate the problem.
If DAB is staining without any ABC, then you need to quench endogenous peroxidase like Baptiste has recommended above (2%). If it doesn't stain with DAB alone, then endogenous peroxidase isn't the problem. If it does stain with ABC/DAB (alone without antibodies) then you have a different problem altogether. If not, then it would indicate your secondary is the problem (as Elizabeth has mentioned). Try buying or borrowing another secondary and see if it works. If not, then one random thing you might try -- and I have no idea if this would work -- is to pre-incubate your secondary antibody with some evans blue (in a small volume like 20ul) to see if it 'blocks' the non-specific staining without adding too much background. Oh, if you are using Ni2+, leave it out and see what happens.
Good luck.
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