Brett Connoly wrote:
I looking for advice on proper fixing/freezing of cell pellets for cryostat
sectioning and subsequent fluorescent IHC and ISH. These will be separate
populations of monocytes, PMNs, eosinophils, and lymphocytes from peripheral
blood and we'll be staining for monos (CD68), PMNs (CD18,) T & B cell
markers, other various cytokines and chemokine receptors. Your
suggestions/secrets and protocols will be greatly appreciated. Should 4%
paraformaldehyde be avoided?
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Dear Brett,
In ISH you can use, in general, aldehyde fixation. Your probe will still
recognize target DNA or RNA. A problem that might arise is accessibility of
the specimen when using a cross-linking fixative like glutaraldehyde.  In
ISH your primary antibody will determine the fixation protocol that can be
used. In my opinion the supplier should include information on fixation
compatibillity in the package insert.
The most convenient way to handle single cells/cell pellets is by embedding
them in gelatin. I always use a concentration of 10 to 20% in PBS. If this
concentration has a negative effect on signal intensity (masking) you have
to go down with the concentration (2-5%).
Embedding must be homogeneous and cells evenly distributed to achive an
optimum effect. After fixation (if this is compatible with your primary)
the cell pellet is brought on top of a 37 oC gelatin/PBS solution and spun
down. Remove as much gelatin as possible. Resuspend to obtain a loosly
packed cell pellet. The gelatin is soldified on ice. Best handling and
sectioning properties are achieved when a second fixation with
(glutar)aldehyde is possible (again depending on primary antibody). Remove
the gelatin/cell pellet from the tube. Optionally the pellet can be
cryoprotected in sucrose, otherwise directly submerged in e.g. LN2.
Hope this is of help, Peter


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Peter van de Plas
AURION
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6702 AA Wageningen
The Netherlands
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