I have been working to optimize an HRP/DAB staining protocol for neuronal reconstructions in my lab for some time, now, and have consistently had issues with staining of distal dendrites. Our protocol does not call for nickel/cobalt enhancement, and I had previously (I think incorrectly) assumed that this was largely to increase contrast, rather than absolute signal.
Is it possible for me to now go back and unmount previous tissue sections (mounted in gelvatol), and enhance existing DAB signal with a heavy metal solution? I'm not clear on whether the metal enhancers interact with HRP (and therefore this probably won't work), or if the metals simply interact/react with the DAB product.
Dear Rogan,
From my experience with DAB reactions, you have to get it right the first time you try it. Efforts afterwards do not improve the outcome, therefore even if you get your sections floating again you will not get any improvement of the staining you already have. The Ni-intensification of DAB reaction using TRIS based buffer is very sensitive and once the tissue stained it will response to subsequent reactions.
For details of my old protocol see my PhD thesis and JCN paper, I have listed for you below. In JCN paper you will see beautiful labelling of distal dendrites and spines (e.g., in cerebellar Purkinje cells).
best wishes, Refik
Thesis Molecular physiology of ATP-gated ion channels assembled fro...
Article Distribution of the P2X2R receptor subunit of the ATP-gated ...
Very much appreciated, thank you! I'll keep your methods in mind as I continue to adjust my protocol. It's worth noting, however, that I'm performing reconstructions of neurobiotin-filled cells, as opposed to immuno. Reading back, I definitely failed to make that clear.
OK I see. I am also doing Neurobiotin fills but I use Streptavidin cy3 to visualise with confocal microscope and Imaris to reconstruct.
best wishes, Refik
Dear Rogan,
I agree with Refik. The best way to enhance the DAB reaction is during the reaction itself, since it is the HRP enzyme that uses the DAB, along with the H2O2, to create the colored product. So it doesn't matter if you are doing immunohistochemistry, detecting neurobiotin in slices, or Western Blots, as long as you have active HRP.
I also vote for Tris-nickle enhancement, and I prefer using Nickle Ammonium Sulfate. In my hands, I risk getting a precipitate with Nickle-Cobalt. If Refik's recipe doesn't work for you, I am happy to send you mine.
While HRP is a pretty hardy enzyme, the only time I have successfully re-reacted sections is before they have dried out or have been cover-slipped. There is the off chance that Gelvatol may have kept the enzyme active, but I wouldn't bother trying unless you really need the data from those sections. However, if you do need the data, you might want to see if someone has recent experience with silver intensification of DAB. I used this in the 1990's, but I used to buy a kit.
Good Luck!
Jill
Hi Jill,
That was my worry, but I may still give the post-staining a shot, just to see. The tissue samples are relatively precious. In any case, I truly appreciate your input!
Cheers,
Rogan
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