Cytokeratin (8/18) -Reply

From:Tony Henwood <>

There could be a problem with the trypsin. Try a section of human liver
and see whether the ducts stain.

Tony Henwood JP, BappSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at  Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: (02) 9845 3306
Fax: (02) 9845 3318

>>> James Mubiru <> 13/June/2001 08:45am >>>
I recently did immunostaining for cytokeratin (8/18)
(NCL-5D3) on mouse prostate tissue. This antibody is
from Novacastra clone 5D3. It is a mouse monoclonal.
All my sections did not stain. I digested the tissue
with 0.4% trypsin for 10 minutes at room
temperature.The trypsin I use is the one from
Sigma(DPCC treated).  I know this antibody is supposed
to stain for glandular epithelium so the prostate
acinar cells should at least stain. The antibody
dilution I used was at 1:100 which is recommended by
the supplier. I incubated the primary antibody for 1
hour at room temperature. I also used the M.O.M kit
from vector since the primary antibody is a mouse
monoclonal and I am detecting in mouse tissue.
My question is if I try to use microwave treatment
with citrate buffer will I get a postive staining?. If
somebody has experience on this antibody on mice I
would appreciate the help. Thank you.
James Mubiru
San Antonio

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