Re: c-kit (CD117)

From:Bryan Hewlett


We have lots of experience with two polyclonal antibodies against
Both work very well on tissues fixed for 24-72 hours in NBF(4% formaldehyde
in Sorenson's phosphate buffer, pH7.0).

1) The rabbit polyclonal(C-19) from Santa Cruz. See the first of the
references below for full technical details and pictures.
This antibody is diluted 1:500 in TBS containing 1% v/v non-immune goat
serum and 10% human serum, incubation is overnight at R.T., detection by
biotinylated goat anti rabbit and streptavidin/peroxidase. HIER is
contra-indicated if the fine dendritic processes of ICC are to be seen.

2) The rabbit polyclonal from Dako. When used on the same tissues,
essentially identical results were obtained. However, this antibody is
supplied already absorbed against human and is much cleaner. Dilution is
1:100 in Dako antibody diluting buffer, incubation again is overnight @RT(
these longer incubations are demonstrably superior for dendritic processes),
detection is the same. We have had three or four cases where HIER improved
the result, but we consider it a last resort.

Choose your control material carefully. I do not recommend using tumor
material, since in tumors, c-kit expression may be either up- regulated or
down- regulated. I would suggest normal jejunum or ileum. Titre your primary
to show very strong staining of mast cells, strong staining of the ICC's and
their dendrites, with weak staining of only some myenteric plexus ganglion
cells. This should give the appropriate staining intensity for GIST,
melanoma, seminoma and other tumors which may show c-kit immunoreactivity.


Sircar, K., Hewlett, B.R., Huizinga, J., Chorneyko, K., Berezin, I., and
Riddell, R.H.  Interstitial cells of Cajal as precursors of gastrointestinal
stromal tumours.  Am. J. Surg. Pathol. 1999; 23(4): 377-389.

Robinson, T.L., Sircar, K., Hewlett, B.R., Chorneyko, K., Riddell, R.H.,
Huizinger, J.D.

Gastrointestinal Stromal Tumors May Originate from a Subset of CD34-Positive
Interstitial Cells of Cajal. Am.J.Pathol 2000; 156(4): 1157-1163.

Please feel free to contact me should you have questions.



----- Original Message -----
Sent: Monday, April 01, 2002 12:03 PM
Subject: c-kit (CD117)

> Anyone using this antibody? I'd be interested in your experience and
> Thanks
> Ronnie Houston
> Regional Histology Operations Manager
> Bon Secours HealthPartners Laboratories
> St Mary's Hospital
> 5801 Bremo Road
> Richmond, VA 23226
> (804) 287 7972
> (804) 288 1008 - fax

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