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BioFX
EoProbe Eosinophil Staining
Kit |
EoProbe is a proprietary label
that reacts with eosinophilic
granular proteins by producing
specific granule protein based
photoreactive complexes. An
eosinophil that has completely
degranulated will not be marked, but
the literature indicates that
complete degranulation almost never
occurs. Partially degranulated cells
show up very well, but the
fluorescence profile looks quite
different and can actually aid in
differentiating activated and
non-activated cells. Eosinophils
are important cells for allergy,
inflammation, tissue rejection and
parasite infections.
Components
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Permeablizing Solution (Reagent A)
PRBS-0025-01
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Probe Solution (Reagent B)
EOPS-0025-01
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Wash Solution (Reagent C)
WSHI-0025-01
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Control Slides -
EOCS-0001-04
Product Use
Histological specimens fixed in 10%
neutral buffered formalin and
cytocentrifuge preparations,
similarly fixed and unfixed can be
used. Cellular preparations can be
used in flow cytometry as well. Histological sections should be deparaffinized
prior to product use. Preparations should
be rehydrated with washing solution
in excess at room temperature for 5
minutes. Specimens should then be permeabilized
with Reagent A for
1 minute at room temperature, in
excess. The slides then should be
probed with probing Reagent B for 1
- 10 minutes (1 minute for thin
specimens, 10 minutes for thick 60
mm specimens). Slides should then be
rinsed with Reagent C and
cleared in running water for 5
minutes. Next, slides should then be
dehydrated in 100% ethanol twice
and washed twice in xylene before
mounting with mounting media.
The control slides supplied in the
kit are cytospins of eosinophils
that will mark positive in this
photocomplex reaction and they are
prepared in a proprietary manner.
Fluorescence should be performed
with an excitation filter of
510-590 nm, dichroic mirror of 580 nm
and a barrier filter of 590 nm. Continuous photoexcitation should be
used, for at least 1 minute, in
order to photobleach all
non-specific autofluorescence. Fluorophore labeled tissues will
provide stable deep red fluorescent
emissions (greater than 605 nm) in eosinophil specific granules in
contrast to a dark background. Four
positive control slides are
included, which consist of cytoprep
specimens containing approximately
20% to 30% eosinophils. These should
be processed similarly as with
"unknown" specimens detailed above.
EoProbe labeled eosinophils in
intestine, digital processed.
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