can you use a nonqueous mounting media like permount (fisher) instead of the aqueous. So do 30seconds each (70%, 95, 100, xylene,xylene) then permount and coverslip. If you need to remove coverslip, you can solubilize the permount with xylene.

Hi,

I use DAB with Nickel intensification on free-floating brain sections, and as Partha mentions, I use DPX as a mounting medium (alcohol & xylene dehydration) and see very little degradation. However, I do see variation between animals so I think things like perfusion/fixation can affect how strong the staining is.

Out of curiosity, how are you quantifying the DAB? I'm doing manual counts, which are so very time consuming! If you have any time-saving tips, that would be great!

Hi I too used DAB with Nickel intensificsation on free-floating brain sections. I followed a similar protocol to Partha and used that method to Immunohistochemically identify c Fos staining . DPX was my mounting medium and had perfect dark black reaction lasted for long time. I think it is a good idea to use dehydration and DPX as suggested by Partha above.

cheers,

Keerthi

Keerthi - I too am doing cFos immunohistochemistry - do you mind if I ask you about quantification? As I mention above, I'm currently doing it manually but have been trying to automate it for some time now!

I have had this issue as well when using DAB with Nickel when staining GFAP positive astrocytes. I used Kaisers to cover slip and I have found if you keep them constantly at four degrees the stain does not fade.

Hi,

I did it manullay using camera lucida drawings of brain sections.

I have found that DAB tends to fade with aqueous mounting media, but had not faced this problem with permount, so I would go with what Partha has suggested.

We usually use very little 100% resin onto gelatin coated slides then cover slips and cure two overnights at 60 especially if you need to draw your profiles under Neurolucida or later examining ultrastructure under TEM from ultrathin sections.

I have experienced disapperance of Ni-intensified DAB with Canada balm within a few months, but never with DPX mounting medium. Following counterstaining with Giemsa stain, brown DAB turn black.

Same here, I also dehydrate and mount with Permount. Black stain is maintained for years, and slides can be kept at room temperature.

Thanks Everyone for your replies-

I know that DPX would have worked right off the bat, however to avoid shrinkage in our tissue investigators were wanting to use an aqueous mounting medium with Nickel enhanced DAB stain. Looks like the metals aren't stable in anything aqueous so a very slow dehydration and mounting with DPX it is!