cytokine IHC

<< Previous Message | Next Message >>
Content-Type:text/plain; charset=us-ascii

Thanks muchly Gail for the explanation.  I do remember the saponin
conversations from a while back.  I guess it is because I'm from Missouri
(the Show-Me State with a Missouri mule as a symbol) that I'm still having
trouble understanding this.

Newly synthesized proteins move from the ER to the cis to  the trans faces
of the Golgi and then to the secretory vesicles for whatever may  their
final destination.  If you are insulin, you might be stored away in
vesicular form or released through the cell membrane to enter the
bloodstream.  If you are an MHC protein, your vesicle  might be fused with
a lysosome to  load the MHC groove with processed antigen which is then
packaged to the outside of the cell membrane.  Whatever your fate, protein
to ER to Golgi story is not unique to cytokines.  And most of these
proteins (besides cytokines) are not freely floating in the cytosol. They
are membrane sequestered.  Yet you can stain them intracellularly without

So I can certainly understand the rationale for saponin when doing FACS
analysis (and I do use it for that) or if the cells are "whole" such as in
a cytospin, but if you are cutting open  (through) a cell when sectioning,
why do you still permeabilize it?
When doing a kappa or lambda stain and you are seeing cytoplasmic staining,
those immunoglobulin subunits are not free in the cytosol but are
sequestered yet you need no saponin.  When staining for insulin in a beta
cell, you can by EM actually see that the insulin is sequestered in
granules yet you need no saponin to stain those endocrine proteins quite
easily.  What is different with cytokines?

My spleen and liver controls from in vivoLPS administration  show very nice
no staining, upregulated staining and then down regulation of staining
depending on the timing of the stimulation and which correlates well with
the dynamics of serum levels of the cytokines (by ELISA) of those same
cytokines.   Use of saponin did not affect these results.

So I'm sorry, dense me, I'm still having trouble understanding why a
sectioned cell, with ER and Golgi randomly cut through and thus exposed,
still needs saponin.

Thanks for any thoughts from anybody.


<< Previous Message | Next Message >>