[Histonet] Artifact in IHC

[Geoff]

Assuming that the 'crust' is a precipitate, what is it soluble in? 
Water, alcohol, dilute acid or base?
It shows up if you just run the peroxide+DAB rxn?

Geoff

On 9/19/2014 8:14 AM, Donna Maney wrote:
> Hello,
> I've been doing IHC on free-floating brain sections for about 15 years using the same protocol: primary raised in rabbit, Vector goat anti-rabbit secondary, Vector ABC Elite kit, then DAB (either nickel-enhanced or not). On the most recent run of tyrosine hydroxylase-labeled tissue we got a lot of weird looking artifact. I am going to upload an image showing the artifact at several magnifications.
>
> - When looking at the slide with the naked eye, the artifact looks like a white crust. Under the scope, it looks black.
> - It appears in patches on the tissue, not over the rest of the slide.
> - It appears in nickel-enhanced (purple, shown in photo) AND regular (brown) DAB stained tissue.
> - It appears even if primary and/or secondary antibody are omitted.
> - It does not always appear in the same area of the section. Some sections do not have it at all.
> - All of the tissue was from the same brain.
>
> Brains were immersion-fixed in 5% acrolein, sunk in 30% sucrose and stored at -80˚ for about a year. They were then cut at 50µm on a freezing sliding microtome and stored in cryoprotectant at -20 for about a week before the IHC. These storage times have not previously been a problem for this IHC.
>
> The IHC was run using 0.01M PBS, pH 7.4.
>
> The stained sections were mounted on gelatin-subbed slides and left to dry overnight. Then they were dehydrated in increasing concentrations of ethanol, then xylenes, then coverslipped in DPX.
>
> Here is the image: http://histosearch.com/imageupload/artifact-in-ihc/
>
> Thanks for any ideas about what this could be.
> Donna
>
>
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Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
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mcauliff <@t> rwjms.rutgers.edu
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