Hi all.  I'm working on a research project in which we
are looking for a cell type called LOX in an animal
model.  (The cells are of human origin and the animal
is mouse.  [They were injected into the mouse.] I know
about the "Alu" in-situ technique for demonstrating
human cells, so no responses leading in that
direction, please...)

OK, so ideally what we'd like to do is find a "QUICK
and dirty" histo stain to do on our tissue.  (NOT
immuno stain or in-situ), and we would like some stain
that is "glaringly" positive (i.e. positive cells
really jump out at you at low power) because we'd also
like to just be able to screen the tissue QUICKLY at
low power.  

My boss had me try a Fontana-Masson's stain.  The
research the tissue came from told us LOX cells are
AMELANOTIC melanoma cells, but my boss seemed to
expect the cells to stain anyway, saying the Fontana
stain would pick up any pro-melanin (melanin
precursors).  I don't know much about this cell line,
I'm thinking the fact they're amelanotic COULD be that
the precursor don't get turned into melanin, but it
also could be the precursors were never there in the
first place.

OK, anyway, I tried a Fontana on these tissues
yesterday, on some sections that we know to contain
LOX cells,(from morphological examination at high
power).  Needless to say it did not work.  I have
since located a source of positive control slides,
they're on their way, so once they're in I'll be able
to double-check my protocol.

SO:

1)Does anybody know anything specifically about LOX
cells & a QUICK way to stain them?

2)Does anybody have any special hints about the
Fontana Stain (whether these cells do turn out to be
Fontana-positive, or not).  My protocol has a Gold
Chloride step, but the one in the text "Theory &
Practic of Histological Techniques" edited by Bancroft
& Stevens DOESN'T have a gold chloride step.  Can
someone tell me what the Gold Chloride is for / what
it is doing.  Also, none of my texts seem to go into
the chemical mechanisms behind this stain, so I'm also
wondering what the sodium thiosulfate step does. 
(Since I don't know anything about the chemistry
behind this stain, I really have no clue which steps I
might want to "play" with the times on to try & get
the stain to work.  Maybe some of the steps are
mordanting steps or others are differentiations?) 
Finally, my protocol said to make the ammoniacal
silver sol'n in the following manner:  To the silver
nitrate sol'n add ammonium hydroxide drop by drop
until the solution clears and no precipitate remains. 
The add the reserved Silver Nitrate Sol'n, drop by
drop, until the solution turns slightly cloudy. 
"Slightly cloudy" sounds like a pretty subjective
concept to me.  Does anyone have any hints on what is
the right "amount" of "cloudiness"?  (Back to the
chemistry questions):  If this sol'n was too cloudy or
not cloudy enough, could that have affected the stain?

By the way, my protocol was:
Ammoniacal Silver Solution 1 hr at 60C
Wash - Distilled water
0.1% Gold Chloride 1 minute
Wash - Distilled water
5% Sodium Thiosulfate 2 minutes
Wash - distilled water
Nuclear Fast Red 5 minutes
Wash
Dehydrate & Clear

ALSO - has anyone ever tried using hematoxylin as a
counterstain for the Fontana?  We don't like Nuclear
Fast Red very much.

OK, thanks for your help!

Sincerely, Miriam Schroeder
Research Associate, Berlex Biosciences

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