Background DAB Precipitate

4 posts / 0 new
Last post
katiemade
katiemade's picture
Background DAB Precipitate

Hi Guys

I am performing DAB:Ni staining in mouse brain cryosections (adult and neonatal). I get a background black precipitate on my sections. In adults it isn't too much of a problem as it seems to be confined to areas that I am less interested in, however in the neonatal sections that I have just started using the precipitate appears across the whole section.

Does anyone know if it is possible to eliminate this background staining? I have outlined my protocol below

I perform an antigen retrieval step, and wash my sections in PBS. I block in normal serum (have used 2% BSA in the past and bg still appears).
The DAB solution is made from sigma tablets and I filter it after it has been dissolved in PBS.

I have attached a picture to show what the bg looks like.

Thanks

samm
samm's picture
How critical is the Ni - i.e.

How critical is the Ni - i.e. are you okay with brown (and a nuclear stain like methyl green) or do you need the black staining?
For the former - eliminate Ni.
If you need black immunostaining, use a new stock of H2O2 and Ni when you make your DAB mix. I usually use the Vectastain system with the 4 bottles, and 'aged' kits often show the Ni ppt, though the DAB seems to work fine.

samm
samm's picture
Also, ensure that your PBS is

Also, ensure that your PBS is free of Fe/Ca/Mg - or just use DI 18 mho water for the DAB.

katiemade
katiemade's picture
Thanks for that.

Thanks for that.

I have used DAB without Ni also and I get the black preciptitate with that as well. i will try the DAB with water instead of PBS as you suggest.