Mary,

We've done some work using different cytokeratin antibodies on murine
tissue.  Wide spectrum cytokeratin has worked very well using formalin fixed
tissues with citrate antigen retrieval.  Cytokeratin 10 works well with NBF
fixed tissue or ethanol fixed tissue.  No retrieval necessary with ethanol
fixed, citrate with NBF.  AE1/AE3 works okay with enzymatic digestion.

Over the past year or so we've been using the DAKO ARK kit for all mouse
monoclonals used on murine tissue.  I've found that in my hands running the
incubation times out to 60min RT and the detection out to 30 RT increases
the intensity.  I'm not sure there is a way to quality the biotinylation
step, to know that enough of your antibody has actually been conjugated to
the biotinylated secondary complex.

I would call the manufacturers of you antibodies and see if they have any
cross reactivity information for cyto 4,8 and 18.  It will save you many a
headache.  Its encouraging to know that someone has been able to show cross
reactivity.

I'm unable to give you any info of  cyto 4, 8 or 18, have yet to give them a
try.  You may be able to add a piece of human or other control tissue to
your ARK run to test success of biotinylation, etc.

Good luck

Tim Coskran

Discovery Pathology
PGRD
Pfizer
860-715-4558



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