Re: Sorting onto slides
David Flowers (dflowers@fred.fhcrc.org)
Thu, 21 Sep 1995 10:26:21 -0800
We have done some sorting of rare CD34+Lin-CD38-Small cells onto slides for
immunoperoxidase staining. All of this was done using the cloner on a BD
Vantage. Since we usually do cloning into 96 well plates, I taped the slide
on top of a plate. At first we were using some special slides called
adhesio slides which I've been told are now only available in Germany. Each
of these slides had about 8 round areas with hydrophobic rings around each
area. We would put about 20-50 lambda of PBS over an area and try to get
500 cells. Then the slides were left in humid cold for an hour or two to
let the cells settle onto the slide. There was some cell loss after the
staining procedure but enough were left for meaningful interpretation.
Later we ran out of the adhesio slides and we started using some home brew
slides treated with 3-amino-propyltriethyloxysaline(APTS) with rings made
using nail polish to contain the PBS. I was told that during the staining
procedure the nail polish came off but not the cells! The reference for the
APTS treatment of glass slides is:
Rentrop,M.,Knapp,H. Winter and J. Schweizer. 1986 Aminoalkylsaline-treated
glass slides as suport for in situ hybridization of keratin cDNAs to frozen
tissue sections under varying fixation and pretreatment conditions.
Hitsochem. J. 18:271-276.
I only did the cloning. All of the slide preparation and immunoperoxidase
staining was done by one of our fellows. She got similar results with both
types of slides.
Hope this helps.
Dave Flowers
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