[Histonet] Artifact in NADH staining

[Yak-Nam Wang]

Hello all,

I have a question regarding some peculiar staining I am getting. Sorry for
the long post.

Goal:
The goal is to identify lesion size by looking at the area of dead cells
using enzyme histochemical analysis.

Experimental details:
Fresh bovine liver (ex vivo) is treated with high intensity focused
ultrasound, to form distinct lesions. Immediately following treatment the
tissue is immediately frozen (embedded in OCT using cooled isopentane). 6-8
um sections are taken and incubated with NADH + nitroblue tetrazolium in
buffer (Ringers and PBS) and then rinsed in DI water before being mounted in
aqueous mounting medium (as described by Neumann RA et al. 1991 "Enzyme
histochemical analysis of cell viability after argon laser-induced
coagulationn necrosis of skin"). Negative control is untreated liver,
positive control is cooked liver.

Results and problem:
Control tissue and untreated tissue stains very nicely. We see a purple
stain localized to the cytoplasm of the cells as expected from the formazan
produced from the reaction. The surrounding ECM is not stained, nor are the
nuclei. The positive control and most regions of the treated tissue also
give expected results- the cells are surrounding areas are unstained.
However, in some areas of the treated tissue (in distinct geometries) we are
seeing this deep blue-black stain around the cells (the cells are not
stained). 

I was wondering what this blue-black stain is. Does anyone have any ideas?
I have seem some reference to a blue-black crystalline precipitate (Novikoff
1959 and Fallon 1974) that form on lipide droplets but haven't been able to
find any further explanation. I don't even know if this is even what I am
seeing as I do not see 'crystals'.

I have just sent a picture to be posted on www.histonet.org I believe it
will take 24 hrs to get it posted on the site. Name is NADH staining
artifact.

We are stumped. Any insight would be greatly appreciated.
Thank you
Yak-Nam