The attached images are at 40x total magnification. (Please excuse the lack of a scale bar; it disappears during file conversion.)
Blocking for endogenous peroxidases does not solve the issue. (To check this, I included a 30 minute incubation in 3% H2O2 before antigen retrieval.)
We use the at-least 98% p.a. 3,3'-Diaminobenzidin Tetrahydrochlorid powder from Carl Roth (Order # CN75.2), and the buffer for our DAB solution is 0.175M sodium acetate with 5% imidazole.
Any suggestions for how to solve this problem would be very much appreciated.
How was the rodent's brain sampled? If you use perfusion techniques, sometimes a subpar saline wash (right before PFA) will leave blood cells and other biological debris in the brain that would normally be washed away by the saline. This will definitely have an impact on the quality of your sections after DAB revelation, even if you control for endogenous peroxidase.
Thank you, Laetitia Rouleau Reduron! The brains were indeed intracardially perfused. Unfortunately/fortunately, I don't think the saline wash is our issue, since previous stainings with the same tissue did not result in any of this dirt. (The samples are from two experiments conducted one year apart from each other [2018 and 2019] and have been stored in antifreeze solution at -20 degrees C). What I will try next is a comparison with different DAB buffers.
filtering is really important! additionally, I find I have to do minimum three rinses in buffer or DI before beginning the dehydration process. I also use the DAB tablets as opposed to a powder; I'm not sure that will make much difference, but you should filter either one before use. good luck!
Thank you, Gail Stonebarger! Those three rinses are part of our protocol also (we do 5 minutes per rinse). I actually did find that tablets work better than the powder! They are just sadly too expensive for the amount of solution our lab regularly uses.
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