[Histonet] Artifact in IHC

Donna Maney dmaney22 <@t> yahoo.com
Fri Sep 19 07:14:45 CDT 2014

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.

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